Cyclic di-AMP induction of type I interferon

ABSTRACT

Methods of modulating type-I interferon production in a cell are provided. Aspects of the methods include modulating cytosolic cyclic di-adenosine monophosphate (c-di-AMP) activity in the cell in a manner sufficient to modulate type-I interferon production in the cell. Additional aspects of the invention include c-di-AMP activity modulatory compositions, e.g., c-di-AMP, mutant  Listeria  bacteria, cyclase and/or phosphodiesterase nucleic acid or protein compositions, etc. The subject methods and compositions find use in a variety of applications, including therapeutic applications.

RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119(e), this application claims priority to U.S.Provisional Patent Application No. 61/423,497 filed on Dec. 15, 2010,the disclosure of which is herein incorporated by reference in itsentirety.

GOVERNMENT RIGHTS

This invention was made with government support under grant nos. P01AI063302 R01 AI027655 and T32 CA009179 awarded by National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Interferons (also referred to as “IFN” or “IFNs”) are proteins having avariety of biological activities, some of which are antiviral,immunomodulating and antiproliferative. They are relatively small,species-specific, single chain polypeptides, produced by mammalian cellsin response to exposure to a variety of inducers such as viruses,polypeptides, mitogens and the like. Interferons protect animal tissuesand cells against viral attack and are an important host defensemechanism. In most cases, interferons provide better protection totissues and cells of the kind from which they have been produced than toother types of tissues and cells, indicating that human-derivedinterferon could be more efficacious in treating human diseases thaninterferons from other species. Interferons may be classified as Type-I,Type-II and Type-III interferons. Mammalian Type-I interferons includeIFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta), IFN-ε(epsilon), IFN-τ (tau), IFN-ω (omega), and IFN-ζ (zeta, also known aslimitin). Interferons have potential in the treatment of a large numberof human cancers since these molecules have anti-cancer activity whichacts at multiple levels. First, interferon proteins can directly inhibitthe proliferation of human tumor cells. The anti-proliferative activityis also synergistic with a variety of approved chemotherapeutic agentssuch as cisplatin, 5FU and paclitaxel. Secondly, the immunomodulatoryactivity of interferon proteins can lead to the induction of ananti-tumor immune response. This response includes activation of NKcells, stimulation of macrophage activity and induction of MHC class Isurface expression leading to the induction of anti-tumor cytotoxic Tlymphocyte activity. In addition, interferons play a role incross-presentation of antigens in the immune system. Moreover, somestudies further indicate that IFN-β protein may have anti-angiogenicactivity. Angiogenesis, new blood vessel formation, is critical for thegrowth of solid tumors. Evidence indicates that IFN-β may inhibitangiogenesis by inhibiting the expression of pro-angiogenic factors suchas bFGF and VEGF. Lastly, interferon proteins may inhibit tumorinvasiveness by affecting the expression of enzymes such as collagenaseand elastase which are important in tissue remodeling.

Interferons also appear to have antiviral activities that are based ontwo different mechanisms. For instance, type I interferon proteins (αand β) can directly inhibit the replication of human hepatitis B virus(“HBV”) and hepatitis C virus (“HCV”), but can also stimulate an immuneresponse which attacks cells infected with these viruses.

The method of administering interferon is an important factor in theclinical application of this important therapeutic agent. Systemicadministration of interferon protein by intravenous, intramuscular orsubcutaneous injection has been most frequently used with some successin treating disorders such as hairy cell leukemia, Acquired ImmuneDeficiency Syndrome (AIDS) and related Kaposi's sarcoma. It is known,however, that proteins in their purified form are especially susceptibleto degradation. In particular, for interferon-β, the primarymechanism(s) of interferon degradation in solution are aggregation anddeamidation. The lack of interferon stability in solutions and otherproducts has heretofore limited its utility. Therefore, a more effectivemethod of modulating the level of interferons, such as interferon-β, isneeded.

SUMMARY

Methods of modulating type-I interferon production in a cell areprovided. Aspects of the methods include modulating cytosolic cyclicdi-adenosine monophosphate (c-di-AMP) activity in the cell in a mannersufficient to modulate type-I interferon production in the cell.Additional aspects of the invention include c-di-AMP activity modulatorycompositions, e.g., c-di-AMP, mutant Listeria bacteria, cyclase and/orphosphodiesterase nucleic acid or protein compositions, etc. The subjectmethods and compositions find use in a variety of applications,including therapeutic applications.

Methods of modulating type-I interferon production in a cell areprovided. Aspects of the methods include modulating cytosolic cyclicdi-adenosine monophosphate (c-di-AMP) activity in the cell in a mannersufficient to modulate type-I interferon production in the cell.Additional aspects of the invention include mutant Listeria bacteriahaving a mutation which modulates secretion of a compound selected fromthe group consisting of: c-di-AMP; cytosolic di-adenylate cyclase andc-di-AMP phosphodiesterase; as compared to its corresponding wild-typecontrol. The subject methods and compositions find use in a variety ofapplications, including therapeutic applications.

Accordingly, aspects of the invention include methods of modulatingtype-I interferon production in a cell by modulating cytosolic cyclicdi-adenosine monophosphate (c-di-AMP) activity in the cell in a mannersufficient to modulate type-I interferon production in the cell.

In some instances, the method is a method of enhancing type-I interferonproduction in a cell and the method comprises increasing cytosolicc-di-AMP activity in the cell. In some of these embodiments, the methodcomprises introducing c-di-AMP or a functional analogue thereof into thecell. In some of these embodiments, the method comprises introducinginto the cell a bacterium that increases cytosolic c-di-AMP in the cell,e.g., a Listeria bacterium. The Listeria bacterium may be a mutant thatsecretes enhanced amounts of c-di-AMP as compared to its correspondingwild-type bacterium. The mutant may have enhanced di-adenylate cyclaseactivity as compared to its corresponding wild-type bacterium, e.g., theListeria bacterium may comprise a Imo2120 mutation. The mutant may havereduced c-di-AMP phosphodiesterase activity as compared to itscorresponding wild-type bacterium, e.g., the Listeria bacterium mayinclude an Imo0052 mutation. In some instances, the bacterium secretes adi-adenylate cyclase into the cell, e.g., Imo2120 or a mutant thereof.In some embodiments, the method includes enhancing cytosolicdi-adenylate cyclase activity in the cell, e.g., by decreasing c-di-AMPphosphodiesterase activity in the cell.

In some embodiments, the method is a method of decreasing type-Iinterferon production in a cell and the method comprises decreasingcytosolic c-di-AMP activity in the cell. Embodiments of such methods mayinclude introducing c-di-AMP activity inhibitor into the cell. In someinstances, the method may include introducing into the cell a bacteriumthat decreases cytosolic c-di-AMP in the cell. The bacterium may be aListeria bacterium. In some instances, the bacterium secretes a c-di-AMPphosphodiesterase into the cell, e.g., Imo0052 or a mutant thereof. Insome embodiments, the method comprises decreasing cytosolic di-adenylatecyclase activity in the cell and/or increasing c-di-AMPphosphodiesterase activity in the cell.

In certain methods of the invention, the cell is a macrophage. Inmethods of the invention, the type-I interferon may be interferon-β. Thecell may be in vitro or in vivo, e.g., part of a multi-cellularorganism, such as a mammal (e.g., human).

Methods of the invention may be methods of expression a heterologousprotein in a subject. Methods of the invention may be methods ofvaccinating a subject. Methods of the invention may be methods oftreating a subject for a disease condition.

Aspects of the invention further include mutant Listeria bacteriumcomprising a mutation which modulates secretion of a compound selectedfrom the group consisting of: c-di-AMP; cytosolic di-adenylate cyclaseand c-di-AMP phosphodiesterase; as compared to its correspondingwild-type control. As such, the mutation may be a mutation that enhancessecretion of c-di-AMP. In such embodiments, the mutation may enhancedi-adenylate cyclase activity in the bacterium, e.g., where the mutationis a Imo2120 mutation. The mutation may decrease c-di-AMPphosphodiesterase activity in the cell, e.g., where the mutation is anImo0052 mutation. The mutation may enhance secretion of cytosolicdi-adenylate cyclase, e.g., where the mutation is a Imo2120 mutation.The mutation may enhance secretion of c-di-AMP phosphodiesterase, e.g.,where the mutation is a Imo0052 mutation.

The Listeria bacterium may be Listeria monocytogenes. The Listeriabacterium may be attenuated. In some instances, the Listeria bacteriumincreases interferon-β production in macrophages as compared to itscorresponding wild-type control. In some instances, the Listeriabacterium decreases interferon-β production in macrophages as comparedto its corresponding wild-type control. The Listeria bacterium mayinclude a heterologous nucleic acid, e.g., where the heterologousnucleic acid is integrated. The heterologous nucleic acid may encode atleast one product, such as an antigen.

Aspects of the invention also include vaccines that include a mutantListeria bacterium, e.g., as described above.

Aspects of the invention also include methods of eliciting or boosting acellular immune response in a subject by administering to the subject aneffective amount of a vaccine, e.g., as described above.

Aspects of the invention also include methods for modulatinginterferon-β production in a mammalian subject by administering to themammalian subject an effective amount of a Listeria bacterium, e.g., asdescribed above. In some instances, the mammalian subject has aneoplastic condition, e.g., cancer. In some instances, the mammaliansubject has a viral infection, e.g., Hepatitis C viral infection. Insome instances, the mammalian subject has multiple sclerosis.

BRIEF DESCRIPTION OF THE FIGURES

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures:

FIG. 1. MDR transport activity is necessary for IFN-β induction duringinfection. An IPTG inducible integration vector containinghexa-histidine tagged wild-type, R109A, or G154C mutant MdrM wasintegrated into the mdrM-strain. (a) Relative expression of MdrM_(His6x)varients in L. monocytogenes membrane fractions in the absence (blackbars) and presence (grey bars) of IPTG was quantified by western blot.(b) Intracellular growth of WT MdrM_(His6x) (closed circles), R109AMdrM_(His6x) (open circles), and G154C MdrM_(His6x) (open squares) inbone marrow derived macrophages (BMMs) in the presence of IPTG. (c)qRT-PCR analysis of IFN-β induction by MdrM variants in the absence(black bars) and presence (grey bars) of IPTG during BMM infection.

FIG. 2. (A) IFN-β production by BMMs in response to solid phase extracts(SPE) of marR-L. monocytogenes supernatants in the presence (black bars)and absence (grey bars) of digitonin. IFN-β activity was measured usingISRE L929 cells that generate luciferase in response to type-I IFNstimulation. Data are mean of biological replicates (N=2). (B) IFN-βactivity by BMMs in response to solid phase extracts of sterile filteredculture supernatants from mdrM-, wild-type (WT), marR-, and tetR::Tn917strains of L. monocytogenes. Negative control consists of digitoninpermeabilizing solution alone (dig). Data are mean±SD (N=2). Datarepresentative of two independent experiments. (C) IFN-β stimulatoryactivity of culture supernatants fractionated using reversed-phase HPLC.Activity measured as in (A). Data are the mean activity of biologicalreplicates (N=2)

FIG. 3. Cyclic di-AMP is an IFN-β activating ligand. (a) Tandem massspectrum resulting from collisionally activated dissociation of thesingly charged positive ion at m/z=659.11 formed from an active fractionof Listeria monocytogenes. (b) Tandem mass spectrum of commerciallyobtained sample of purified c-di-AMP. The fragment ions at m/z=641.10and 312.05 correspond to neutral losses of 18 Da from the precursor ion(m/z=659.11) and from the fragment ion at m/z=330.06, respectively, andare consistent with neutral loss of water molecules from theserespective ions. Fragmentation pathways of c-di-AMP are shown in (c).(d) Commercial c-di-AMP standard (BioLog Life Sciences Institute,Denmark) was added to BMMs in increasing amount. IFN-β production byBMMs was detected using the type I IFN reporter cell line (ISRE L929).Commercial c-di-AMP standard and the active L. monocytogenes fractionwere treated with snake venom phosphodiesterase (SVPD). Error barsrepresent standard deviation of single samples measured in triplicate.

FIG. 4. The di-adenylate cyclase gene dacA (Imo2120) alters CSPactivation during infection. (a) Predicted operon of genes Imo2120,renamed here dacA, and Imo2119. The product of each gene is predicted tocontain two transmembrane spanning segments (TM). The gene product ofImo2119 contains three ybbR domains of unknown function. The geneproduct of Imo2120 contains a single di-adenylate cyclase (DAC) domain.(b) Intracellular growth curves of WT L. monocytogenes (closed circles)and L. monocytogenes with an integration vector (pLIV2) containing IPTGinducible dacA in the absence (open circles) and presence (open squares)of IPTG (1 mM) in BMMs. (c) qRT-PCR analysis of IFN-β induction by eachstrain in BMMs.

FIG. 5. Western blot of membrane fraction from the mdrM-L. monocytogenesstrain integrated with an IPTG inducible plasmid containing WT, R109A,and G154C variants of hexa-histidine tagged MdrM. A total of 75 μg ofmembrane protein were separated by SDS-PAGE gel electrophoresis andblotted onto PVDF membrane. Blots were probed with a primaryanti-histidine antibody and a secondary antibody conjugated to aninfrared emitting fluorophore (Licor). Blots were imaged using theOdyssey Infrared Imaging System. The IPTG inducible, anti-His(6×)cross-reactive band at 48.8 kDa was quantified using the accompanyingsoftware.

FIG. 6. (a) Absorbance spectrum from tetR::Tn917 L. monocytogenes HPLCfraction 22 (λ_(max)=260 nm). (b) Active fraction from marR-L.monocytogenes were mixed with (grey bars) and without (black bars) anionand cation exchange resins. Resin was removed by centrifugation and thesupernatant was tested for IFN-β stimulatory activity with BMMs and ISREcells. (c) Digested cell wall from L. monocytogenes in the presence(grey bars) and absence (black bars) of DNAse (PGN digest) was deliveredto macrophage cytosol using lipofectamine 2000. Solid phase extract frommarR-L. monocytogenes (L.m. extract) was treated similarly and IFN-βstimulatory activity was measured as in FIG. 1 a. Error bars representstandard deviations of single samples measured in triplicate.

FIG. 7. Identification of c-di-AMP from Listeria monocytogenes. (a)Isotopically resolved electrospray ionization mass spectrum measured inthe positive ion mode for a fraction of Listeria monocytogenes, showingdetail for the range m/z=658-662. (b) Isotopic distribution calculatedfor the (M+H)⁺ ion of c-di-AMP (M=C₂₀H₂₄N₁₀O₁₂P₂). The simulatedspectrum of (b) was calculated from the natural abundances of theisotopes using Xcalibur software (version 4.1, Thermo).

FIG. 8. Correlation between IFN-β inducing activity of L. monocytogenessupernatants and [c-di-AMP] in these supernatants, as measured by massspectroscopy. IFN-β induction was measured using an interferon bioassaywith ISRE L929 cells. Concentration of c-di-AMP was determined by massspectrometry in comparison with a synthetic standard of knownconcentration. Line represents linear regression fit to data. Equationof fit above the graph.

FIG. 9. Cytosolic detection of L. monocytogenes vs. c-di-AMP. Type-Iinterferon was measured in response to the tetR::Tn917 strain of L.monocytogenes, c-di-AMP, and LPS in (A) WT (black bars) andmyd88-trif−/− (grey bars), (B) WT (black bars) and irf3−/− (grey bars),and (C) mavs+/− (black bars) and mavs−/− (grey bars) macrophages. L.monocytogenes infection was performed at an MOI of 4. Digitonin was usedto deliver c-di-AMP to the cytosol. LPS was used as a positive control.Type-I IFN was measured using the ISRE L929 cells from supernatants ofBMMs after 6 hours.

DETAILED DESCRIPTION

Methods of modulating type-I interferon production in a cell areprovided. Aspects of the methods include modulating cytosolic cyclicdi-adenosine monophosphate (c-di-AMP) activity in the cell in a mannersufficient to modulate type-I interferon production in the cell.Additional aspects of the invention include c-di-AMP activity modulatorycompositions. The subject methods and compositions find use in a varietyof applications, including therapeutic applications.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating recited number may be a numberwhich, in the context in which it is presented, provides the substantialequivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be constructed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Methods

As summarized above, methods of modulating type-I interferon productionin a cell, e.g., in vitro or in vivo, where the target cell is aeukaryotic cell, are provided. By modulating type-I interferonproduction is meant that the subject methods change, e.g., enhance orinhibit, type-I interferon production in a cell, as compared to acontrol. The magnitude of the modulation may vary, and in some instancesis 2-fold or greater, such as 5-fold or greater, including 10-fold orgreater, as compared to a suitable control. As such, in some instances,the methods are methods of increasing type-I interferon production in acell, e.g., by a magnitude of 2-fold or greater, such as 5-fold orgreater, including 10-fold or greater, as compared to a suitablecontrol. In yet other instances, the methods are methods of decreasingtype-I interferon production in a cell, e.g., by a magnitude of 2-foldor greater, such as 5-fold or greater, including 10-fold or greater, ascompared to a suitable control.

Aspects of the methods include modulating cytosolic cyclic di-adenosinemonophosphate (c-di-AMP) activity in the cell in a manner sufficient tomodulate type-I interferon production in the cell. Cytosolic cyclicdi-adenosine monophosphate (c-di-AMP) activity refers to the amount oflevel of active c-di-AMP in a cell. By modulating c-di-AMP activity ismeant that the subject methods change, e.g., enhance or inhibit,c-di-AMP activity in a cell, as compared to a control. The magnitude ofthe modulation may vary, and in some instances is 2-fold or greater,such as 5-fold or greater, including 10-fold or greater, as compared toa suitable control. As demonstrated in the Experimental Section below,the amount of active c-di-AMP in the cell is directly proportional tothe level of type-I interferon production in the cell. As such, in someinstances, the methods are methods of increasing c-di-AMP activity in acell, e.g., as described below, such as 5-fold or greater, including10-fold or greater, as compared to a suitable control. In yet otherinstances, the methods are methods of decreasing c-di-AMP activity in acell, e.g., as described below, by a magnitude of 2-fold or greater,such as 5-fold or greater, including 10-fold or greater, as compared toa suitable control.

Modulation of cytosolic c-di-AMP activity may be accomplished using avariety of different approaches. In some instances, the method comprisescontacting a target cell with a c-di-AMP activity modulatory agent,i.e., an agent that enhances or decreases c-di-AMP activity in thetarget cell. c-di-AMP activity modulatory agents may vary, and includebut are not limited to: small molecules agents, bacterial agents, andnucleic acid/protein agents, etc.

In some instances, the c-di-AMP activity modulatory agent is c-di-AMP ora functional analogue thereof. c-di-AMP has the structure shown in FIG.3 c. Also of interest are functional analogues of c-di-AMP, e.g.,2′-bis(tert-butyldimethylsilyl)-c-di-AMP or as described below, wherethe functional analogues exhibit similar functional activity and mayhave a similar structure to c-di-AMP. In some instances, the functionalanalogue is not c-di-GMP. Of interest as functional analogues are smallmolecule agents. Naturally occurring or synthetic small moleculecompounds of interest include numerous chemical classes, such organicmolecules, including small organic compounds having a molecular weightof more than 50 and less than about 2,500 daltons. Candidate agentscomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, preferably at least twoof the functional chemical groups. The candidate agents may comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.Such molecules may be identified, among other ways, by employingsuitable screening protocols. c-di-AMP and functional analogues findinguse in embodiments of the invention (as well as methods of theiradministration) are further described in published United StatesApplication No. 20080286296; the disclosure of which is hereinincorporated by reference.

Also of interest as c-di-AMP activity modulatory agents are bacterialagents, e.g., a Listeria bacterial agents. Bacteria of interest includemutant bacteria, e.g., mutant Listeria bacteria, which include amutation that modulates secretion of one or more compounds thatultimately modulate c-di-AMP activity in the target cell. Compounds ofinterest whose secretion may be modulated (as compared to a control)include compounds selected from the group consisting of: c-di-AMP;cytosolic di-adenylate cyclase and c-di-AMP phosphodiesterase andcombinations thereof. One or more of these compounds may be modulated ascompared to its corresponding wild-type control, as desired.

In some instances, the Listeria bacteria modulate interferon-βproduction, e.g., in macrophages. The term “modulates” as used hereinrefers to an increase or a decrease, e.g., in secretion of c-di-AMP, ininterferon-β production, etc. In some embodiments, the modulation is anincrease or decrease of at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, or at least about 100% as compared to a Listeria bacteriathat does not include the mutation, e.g., a corresponding wild typecontrol.

In some embodiments, the mutant Listeria bacteria increase interferon-βproduction as compared to Listeria bacteria that do not include themutation, such as a wild-type Listeria bacterium. In such embodiments,the increase is from about 1.5-fold increase to about 50-fold increaseor more, including about 2-fold increase to about 45-fold increase,about 5-fold increase to about 40-fold increase, about 10-fold increaseto about 35-fold increase, about 15-fold increase to about 30-foldincrease, about 20-fold increase to about 30-fold increase, and thelike.

In other embodiments, the mutant Listeria bacteria decrease interferon-βproduction as compared to Listeria bacteria that do not include themutation. In such embodiments, the increase is from about 1.5-folddecrease to about 50-fold decrease or more, including about 2-folddecrease to about 45-fold decrease, about 5-fold decrease to about40-fold decrease, about 10-fold decrease to about 35-fold decrease,about 15-fold decrease to about 30-fold decrease, about 20-fold decreaseto about 30-fold decrease, and the like.

In certain embodiments, mutant species according to the subjectinvention, are ones that modulate (e.g., increase or decrease)interferon-β production as compared to their corresponding wild-typestrain in a macrophage cell culture. In this assay, macrophages areinfected with test and reference, e.g., wild-type, strains of bacteria.Following a period of time, e.g., 4 to 18 hours, the macrophage culturemedia is collected and the amount of Type I interferon secreted by themacrophages is detected using a reporter gene such as luciferase clonedunder regulation of a Type I interferon signaling pathway. The level ofthe reporter gene is then measured test and reference, e.g., wild-type,strains of bacteria to identify mutant Listeria strains that modulate(e.g., increase or decrease) interferon-β production.

The subject bacteria may be any species that includes a mutationaccording to the subject invention. Thus, strains of Listeria other thanL. monocytogenes may be used for the generation of mutants according tothe present invention. In certain embodiments, the Listeria strain is L.monocytogenes.

Specific mutant Listeria bacteria of interest include those that exhibitenhanced production and/or secretion of c-di-AMP by the bacteria, orthat promote enhanced production or activity of c-di-AMP in the cell.Examples of such species include those in which the mutation enhancesdi-adenylate cyclase activity in the bacterium or the cell, e.g., amutation that enhances the expression, secretion or activity of apolypeptide comprising a di-adenylate cyclase, or DAC, domain. By “DACdomain” it is meant a peptide domain that confers di-adenylate cyclaseactivity on a protein, for example, the DAC domain at residues 100-240of the di-adenylate cyclase gene Imo2120 (also known as dacA or YbbP;e.g. Listeria monocytogenes Imo2120, the polypeptide sequence for whichis

(SEQ ID NO: 11)MDFSNMSILHYLANIVDILVVWFVIYKVIMLIRGTKAVQLLKGIFIIIAVKLLSGFFGLQTVEWITDQMLTWGFLAIIIIFQPELRRALETLGRGNIFTRYGSRIEREQHHLIESIEKSTQYMAKRRIGALISVARDTGMDDYIETGIPLNAKISSQLLINIFIPNTPLHDGAVIIKGNEIASAASYLPLSDSPFLSKELGTRHRAALGISEVTDSITIVVSEETGGISLTKGGELFRDVSEEELHKILLKELVTVTAKKPSIFSKWKGGKSE,and the nucleic acid sequence for which is

(SEQ ID NO: 12)ATGGATTTTTCCAATATGTCGATATTGCATTATCTAGCAAATATTGTAGATATTCTTGTCGTATGGTTTGTAATTTATAAAGTGATCATGTTAATCCGAGGTACAAAAGCAGTACAATTACTAAAAGGCATTTTTATTATCATTGCAGTCAAACTATTAAGCGGATTTTTTGGTCTCCAAACAGTAGAATGGATTACGGATCAGATGCTTACTTGGGGATTCCTTGCAATTATAATTATCTTCCAACCGGAATTACGCCGTGCTTTAGAAACGCTTGGACGAGGTAATATTTTTACTCGTTATGGATCAAGAATTGAGCGTGAACAGCATCATTTAATCGAGTCTATCGAAAAATCCACCCAATATATGGCAAAACGTCGAATTGGGGCACTGATTTCAGTGGCACGCGATACAGGCATGGACGATTATATTGAAACAGGTATTCCGTTAAATGCAAAAATTTCTTCTCAATTATTAATTAATATTTTTATTCCGAATACACCGCTTCATGATGGAGCAGTTATTATTAAAGGAAACGAAATTGCATCGGCAGCAAGTTACTTGCCACTTTCAGATAGCCCGTTCTTATCCAAAGAACTTGGAACGCGTCACCGGGCTGCACTTGGGATTAGTGAAGTGACAGATAGTATTACGATTGTAGTTTCTGAAGAGACTGGCGGAATTTCCCTAACTAAAGGTGGAGAACTTTTCCGTGATGTGTCAGAAGAAGAGTTACATAAAATTCTTCTTAAAGAACTAGTCACAGTAACTGCAAAGAAACCTTCTATCTTTTCTAAATGGAAAGGAGGCAAAAGCGAATGA);or the DAC domain at residues 1-145 of DisA (e.g., Thermotoga maritimeDisA, Witte et al. Mol. Cell 30:167-178, the polypeptide sequence forwhich is

(SEQ ID NO: 13)MGVKSLVPQELIEKIKLISPGTELRKALDDIINANFGALIFLVDDPKKYEDVIQGGFWLDTDFSAEKLYELSKMDGAIVLSEDITKIYYANVHLVPDPTIPTGETGTRHRTAERLAKQTGKVVIAVSRRRNIISLYYKNYKYVVNQVDFLISKVTQAISTLEKYKDNFNKLLSELEVLELENRVTLADVVRTLAKGFELLRIVEEIRPYIVELGEEGRLARMQLRELTEDVDDLLVLLIMDYSSEEVEEETAQNILQDFITRREPSPISISRVLGYDVQQAAQLDDVLVSARGYRLLKTVARIPLSIGYNVVRMFKTLDQISKASVEDLKKVEGIGEKRARAISESISSLKHRKTSE).

For example, the bacterial genome may be manipulated to overexpress thenative di-adenylate cyclase gene Imo2120, or a mutant thereof, e.g. afragment or mutant comprising di-adenylate cyclase activity, forexample, by expressing multiple copies of the Imo2120 gene ordi-adenylate cyclase active Imo2102 mutant or expressing the native dacAgene or di-adenylate cyclase active mutant under control of a strong,e.g., constitutive, promoter. By “native” and “native polypeptide” it ismeant a polypeptide found in nature. By “mutant” or “variant” it ismeant a mutant of the native polypeptide having less than 100% sequenceidentity with the native sequence. For example, variants includepolypeptides having 60% sequence identity or more with a full lengthnative protein having di-adenylate cyclase activity, e.g. dacA/Imo2120m,DisA, etc., e.g. 65%, 70%, 75%, or 80% or more identity, such as 85%,90%, or 95% or more identity, for example, 98% or 99% identity with thefull length native protein, where mutants of interest include deletion,insertion and substitution mutants. Variants also include fragments of anative polypeptide, where the variants maintain di-adenylate cyclaseactivity, e.g. the DAC domain of Imo2120, DisA, etc. e.g. a fragmentcomprising residues 100-240 of Imo2120 or the comparable sequence in aImo2120 homolog or ortholog, or comprising residues 1-147 of DisA, orthe comparable sequence in a DisA homolog or ortholog, as well as activemutants thereof. Examples of such species further include bacteriahaving additional active coding sequences of wild type or functionalImo2120, e.g., where the bacteria harbor one or more additional wildtype sequences under the control of a constitutive or induciblepromoter.

Other examples of mutant Listeria bacteria that exhibit enhancedproduction and/or secretion of c-di-AMP by the bacteria or that promoteenhanced production or activity of c-di-AMP in the cell include bacteriathat exhibit reduced cyclic nucleotide phosphodiesterase activity, i.e.,reduced c-di-AMP phosphodiesterase activity, e.g., bacteria comprising anull or hypomorphic mutation in the Listeria gene Imo0052. The aminoacid sequence of Imo0052 is:

(SEQ ID NO: 14)MSGYFQKRMLKYPLYGLIAATIILSVITFFFSWWLSALVVVGGIILTVAMFYFEYRLNEDVQLYVSNLTYRIKRSEEEALVEMPMGILLYDEHYKIEWVNPFMSKYFDKAELIGESLEEVGPEFLDVITGNDEKGIMSIAWRDHRFDTIVKRKERILYLYDRTEYYDLNKKFQANKSVFAVIFLDNYDEWAQGMDDRRRSALNNLVTSMLTNWAREHRIYLKRISTDRFMAFLTEEMLKRLEEEKFQILDRIRERTSKQNIPLTLSIGIGYKEDDLIQLADLAQSSLDLALGRGGDQVVIKQPEGKVRFYGGKTNPMEKRTRVRARVISQALQELITQSDQVFVMGHRYPDMDVIGSSLGVMRIAEMNDRNAYVVVEPGKMSPDVKRLMNEIEEYPNVIKNIVTPQVALENITEKSLLVVVDTHKPSMVINKELLDSATNVVVVDHHRRSEEFVGSPVLVYIEPYASSTAELITELFEYQPDLEQVGKIEATALLSGIVVDTKNFTLRTGSRTFDAASYLRSLGADTILVQQFLKEDITTFTQRSRLVESLEIYHDGMAIATGHEDEEFGTVIAAQAADTMLSMEGVQASFVITLRPDKLIGISARSLGQINVQVIMEKLGGGGHLSNAATQLKDVTIAEAEKQLISAIDAYWKGETThe nucleic acid sequence of Imo0052 is:

(SEQ ID NO: 15)ATGTCAGGCTATTTTCAAAAACGAATGCTTAAATATCCATTATACGGTCTGATTGCAGCGACAATTATTTTGAGCGTAATCACGTTCTTTTTTTCGTGGTGGTTATCGGCGTTAGTTGTTGTTGGCGGAATTATTCTTACGGTTGCGATGTTTTATTTTGAATATCGCTTGAATGAAGATGTGCAACTATATGTTTCTAATTTAACGTATCGGATTAAGCGTAGTGAAGAAGAAGCGCTTGTTGAAATGCCGATGGGAATACTGCTGTATGATGAACATTACAAAATCGAATGGGTTAACCCGTTTATGTCAAAATACTTTGATAAGGCAGAGTTAATCGGGGAATCTTTGGAAGAAGTAGGACCGGAATTTTTGGACGTTATTACTGGGAATGATGAAAAGGGGATTATGTCGATTGCTTGGCGTGATCACCGTTTTGATACGATAGTAAAGCGTAAGGAACGAATTTTATATTTATATGATCGCACAGAATATTATGATTTAAACAAGAAATTTCAAGCGAATAAATCTGTATTTGCGGTTATTTTCTTAGATAATTATGATGAATGGGCGCAGGGCATGGATGATAGACGTCGCAGTGCTTTAAATAATTTGGTGACGTCGATGTTGACCAACTGGGCTAGGGAACATCGTATTTATTTGAAACGGATTTCGACAGACCGATTTATGGCCTTTTTGACGGAGGAAATGTTGAAGCGGTTGGAGGAAGAGAAGTTTCAAATATTGGACCGGATTCGCGAACGGACGTCGAAGCAAAATATTCCTTTAACGCTTAGTATTGGGATTGGTTATAAGGAAGATGATTTGATTCAGCTGGCCGATTTGGCGCAGTCTAGTCTAGATCTTGCTTTAGGGCGCGGCGGCGATCAGGTTGTAATTAAGCAACCTGAAGGAAAAGTGCGTTTTTATGGTGGGAAAACAAATCCGATGGAAAAACGGACTCGTGTTCGCGCGCGTGTGATTTCGCAAGCATTGCAAGAGCTGATTACGCAAAGTGACCAAGTTTTTGTTATGGGGCACCGCTATCCGGATATGGACGTAATTGGTTCGAGTCTTGGAGTGATGCGGATTGCTGAGATGAATGATCGGAATGCTTATGTGGTTGTGGAACCTGGCAAAATGAGTCCAGATGTGAAGCGACTAATGAATGAAATTGAAGAATATCCGAATGTAATTAAAAATATTGTTACACCGCAAGTCGCACTGGAAAATATCACGGAGAAGAGTTTGCTCGTTGTTGTTGATACACACAAACCTTCGATGGTTATTAATAAGGAATTGCTGGACTCAGCTACGAATGTGGTTGTTGTCGATCATCACCGTCGTTCAGAGGAATTTGTTGGGAGTCCGGTTCTTGTTTATATCGAGCCATATGCGTCATCTACTGCCGAATTGATTACGGAGCTATTTGAGTATCAACCGGATTTAGAGCAGGTTGGGAAAATCGAGGCAACGGCGCTTCTTTCCGGGATTGTGGTTGATACGAAGAACTTTACGCTGCGGACTGGGTCGCGAACGTTTGATGCGGCAAGTTATTTACGGTCGCTTGGTGCGGACACGATTTTGGTGCAGCAATTTTTGAAAGAAGATATTACTACTTTTACACAGCGGAGTCGTTTAGTGGAGTCGCTTGAAATTTATCATGATGGTATGGCGATTGCGACTGGACATGAGGACGAGGAATTTGGCACAGTTATAGCTGCGCAGGCGGCAGATACGATGCTTTCGATGGAAGGCGTGCAGGCATCCTTTGTTATTACGCTACGTCCGGATAAATTAATCGGGATTAGCGCGAGATCGCTTGGCCAAATCAATGTGCAAGTCATTATGGAAAAACTAGGCGGTGGCGGACATTTATCGAATGCAGCCACACAGCTTAAAGATGTTACAATTGCAGAAGCAGAAAAACAATTAATTAGCGCCATTGATGCGTATTGGAAGGGAGAAACATAAThe Imo0052 mutant product may differ from the above sequence by 1 ormore residues, where mutants of interest include deletion, insertion andsubstitution mutants.

Instead of or in addition to having enhanced secretion of c-di-AMP,mutant bacteria of interest also include bacteria that exhibit enhancedsecretion of a di-adenylate cyclase and/or decreased secretion of ac-di-AMP phosphodiesterase. See, for example, US Application Pub. No.2010/0285067, the disclosure of which is incorporated herein byreference. Accordingly, also of interest are Listeria bacteria whereinthe mutation increases secretion of cytosolic di-adenylate cyclase,e.g., where the mutation increases the expression of multidrug effluxpumps (MDRs) in the bacteria, e.g. as described in the examples sectionbelow. Also of interest are Listeria bacteria comprising mutations thatdecrease secretion of c-di-AMP phosphodiesterase, e.g., bacteria thatexpress transport-inactive MDR mutants, e.g. as described in theexamples section below.

As mentioned above, mutant bacteria that result in decreased IFNproduction are also interest. Such bacteria may be employed for a numberof different applications, including the delivery of vectors (e.g.,viral vectors, plasmids, etc) or other material into the cytosol of atarget cell. Bacteria of these embodiments may have reduced cyclaseactivity, e.g., they may include an Imo2120 nucleic acid mutation thatresults in expression of a product that has reduced activity as comparedto wild type, or no activity, and or enhanced phosphodiesteraseactivity, e.g., they may overexpress Imo0052, or include an Imo0052nucleic acid mutation that results in expression of a product that hasgreater activity as compared to wild type. For these applications, wheredesirable the bacteria may be strains that lyse in the cytosol, e.g., byexpressing a phage holin and/or lysin.

The above-mutant bacteria may be fabricated using a variety of differentprotocols. As such, generation of the subject mutant bacteria may beaccomplished in a number of ways that are well known to those of skillin the art, including deletion mutagenesis, insertion mutagenesis, andmutagenesis which results in the generation of frameshift mutations,mutations which effect premature termination of a protein, or mutationof regulatory sequences which affect gene expression. Where desired,expression may be mediated by a viral vector. Mutagenesis can beaccomplished using recombinant DNA techniques or using traditionalmutagenesis technology using mutagenic chemicals or radiation andsubsequent selection of mutants. Representative protocols of differentways to generate mutant bacteria according to the present invention areprovided in the Experimental Section, below.

In certain embodiments, the mutant Listeria bacteria are killed butmetabolically active (KBMA). By the term “KBMA” or “killed butmetabolically active” is meant that the bacteria are attenuated forentry into non-phagocytic cells and attenuated with respect tocell-to-cell spread resulting in bacteria that have greatly reducedtoxicity and yet the immunogenicity of the bacteria is maintained. Suchmutants include, but are not limited to, mutations in one or all uvrgenes, i.e. uvrA, uvrB, uvrC, and uvrD genes as well as recA genes, orfunctionally equivalent genes, depending on the genus and species of themicrobe. These mutations result in attenuation in the activity of thecorresponding enzymes UvrA (an ATPase), UvrB (a helicase), UvrC (anuclease), UvrD (a helicase II) and RecA (a recombinase). These mutantswould typically be used in conjunction with a crosslinking compound,such as a psoralen. In one embodiment, there are attenuating mutations,such as deletions, in both uvrA and uvrB (uvrAB). KBMA mutations arefurther described in Brockstedt et al., Nature Med. 11, 853-860 (2005)and in U.S. Published Patent Application No. 2004/0228877.

In certain embodiments, the mutant Listeria bacteria are alsoattenuated. By the term “attenuation,” as used herein, is meant adiminution in the ability of the bacterium to cause disease in ananimal. In other words, the pathogenic characteristics of the attenuatedListeria strain have been lessened compared with wild-type Listeria,although the attenuated Listeria is capable of growth and maintenance inculture. Using as an example the intravenous inoculation of Balb/c micewith an attenuated Listeria, the lethal dose at which 50% of inoculatedanimals survive (LD50) is preferably increased above the LD50 ofwild-type Listeria by at least about 10-fold, more preferably by atleast about 100-fold, more preferably at least about 1,000 fold, evenmore preferably at least about 10,000 fold, and most preferably at leastabout 100,000-fold. An attenuated strain of Listeria is thus one whichdoes not kill an animal to which it is administered, or is one whichkills the animal only when the number of bacteria administered is vastlygreater than the number of wild type non-attenuated bacteria which wouldbe required to kill the same animal. An attenuated bacterium should alsobe construed to mean one which is incapable of replication in thegeneral environment because the nutrient required for its growth is notpresent therein. Thus, the bacterium is limited to replication in acontrolled environment wherein the required nutrient is provided. Theattenuated strains of the present invention are thereforeenvironmentally safe in that they are incapable of uncontrolledreplication.

In certain embodiments, the attenuated mutant Listeria bacteriaaccording to the subject invention are ones that exhibit a decreasedvirulence compared to their corresponding wild type strain in theCompetitive Index Assay as described in Auerbach et al., “Development ofa Competitive Index Assay To Evaluate the Virulence of Listeriamonocytogenes actA Mutants during Primary and Secondary Infection ofMice,” Infection and Immunity, September 2001, p. 5953-5957, Vol. 69,No. 9. In this assay, mice are inoculated with test and reference, e.g.,wild-type, strains of bacteria. Following a period of time, e.g., 48 to60 hours, the inoculated mice are sacrificed and one or more organs,e.g., liver, spleen, are evaluated for bacterial abundance. In theseembodiments, a given bacterial strain is considered to be less virulentif its abundance in the spleen is at least about 50-fold, or more, suchas 70-fold or more less than that observed with the correspondingwild-type strain, and/or its abundance in the liver is at least about10-fold less, or more, such as 20-fold or more less than that observedwith the corresponding wild-type strain. In yet other embodiments,bacteria are considered to be less virulent if they show abortivereplication in less than about 8 hours, such as less than about 6 hours,including less than about 4 hours, as determined using the assaydescribed in Jones and Portnoy, Intracellular growth of bacteria.(1994b) Methods Enzymol. 236:463-467. In yet other embodiments, bacteriaare considered to be attenuated or less virulent if, compared towild-type, they form smaller plaques in the plaque assay employed in theExperimental Section, below, where cells, such as murine L2 cells, aregrown to confluency, e.g., in six-well tissue culture dishes, and theninfected with bacteria. Subsequently, DME-agar containing gentamicin isadded and plaques are grown for a period of time, e.g., 3 days. Livingcells are then visualized by adding an additional DME-agar overlay,e.g., containing neutral red (GIBCO BRL) and incubated overnight. Insuch an assay, the magnitude in reduction in plaque size observed withthe attenuated mutant as compared to the wild-type is, in certainembodiments, 10%, including 15%, such as 25% or more.

In certain embodiments, the subject bacteria are cytotoxic. A particularstrain of bacteria is considered to be cytotoxic if it compromises itshost cell in a period of less than about 8 hours, sometimes less thanabout 6 hours, e.g., in less than about 5 hours, less than about 4hours, less than about 3 hours, less than about two hours, or less thanabout 1 hour, as determined using the cytotoxicity assay describedbelow. In some instances, the strains may induce pyroptosis.Alternatively, or in addition, strains of the invention may becoadministered with other strains, e.g., strains that induceinflammasome activation.

In certain embodiments, mutant bacteria according to the subjectinvention express a heterologous antigen. The heterologous antigen is,in certain embodiments, one that is capable of providing protection inan animal against challenge by the infectious agent from which theheterologous antigen was derived, or which is capable of affecting tumorgrowth and metastasis in a manner which is of benefit to a hostorganism. Heterologous antigens which may be introduced into a Listeriastrain of the subject invention by way of DNA encoding the same thusinclude any antigen which when expressed by Listeria serves to elicit acellular immune response which is of benefit to the host in which theresponse is induced. Heterologous antigens therefore include thosespecified by infectious agents, wherein an immune response directedagainst the antigen serves to prevent or treat disease caused by theagent. Such heterologous antigens include, but are not limited to,viral, bacterial, fungal or parasite surface proteins and any otherproteins, glycoproteins, lipoprotein, glycolipids, and the like.Heterologous antigens also include those which provide benefit to a hostorganism which is at risk for acquiring or which is diagnosed as havinga tumor that expresses the said heterologous antigen(s). The hostorganism is preferably a mammal and most preferably, is a human.

By the term “heterologous antigen,” as used herein, is meant a proteinor peptide, a lipoprotein or lipopeptide, or any other macromoleculewhich is not normally expressed in Listeria, which substantiallycorresponds to the same antigen in an infectious agent, a tumor cell ora tumor-related protein. The heterologous antigen is expressed by astrain of Listeria according to the subject invention, and is processedand presented to cytotoxic T-cells upon infection of mammalian cells bythe strain. The heterologous antigen expressed by Listeria species neednot precisely match the corresponding unmodified antigen or protein inthe tumor cell or infectious agent so long as it results in a T-cellresponse that recognizes the unmodified antigen or protein which isnaturally expressed in the mammal. In other examples, the tumor cellantigen may be a mutant form of that which is naturally expressed in themammal, and the antigen expressed by the Listeria species will conformto that tumor cell mutated antigen. By the term “tumor-related antigen,”as used herein, is meant an antigen which affects tumor growth ormetastasis in a host organism. The tumor-related antigen may be anantigen expressed by a tumor cell, or it may be an antigen which isexpressed by a non-tumor cell, but which when so expressed, promotes thegrowth or metastasis of tumor cells. The types of tumor antigens andtumor-related antigens which may be introduced into Listeria by way ofincorporating DNA encoding the same, include any known or heretoforeunknown tumor antigen. In other examples, the “tumor-related antigen”has no effect on tumor growth or metastasis, but is used as a componentof the Listeria vaccine because it is expressed specifically in thetissue (and tumor) from which the tumor is derived. In still otherexamples, the “tumor-related antigen” has no effect on tumor growth ormetastasis, but is used as a component of the Listeria vaccine becauseit is selectively expressed in the tumor cell and not in any othernormal tissues. The heterologous antigen useful in vaccine developmentmay be selected using knowledge available to the skilled artisan, andmany antigenic proteins which are expressed by tumor cells or whichaffect tumor growth or metastasis or which are expressed by infectiousagents are currently known. For example, viral antigens which may beconsidered as useful as heterologous antigens include but are notlimited to the nucleoprotein (NP) of influenza virus and the gag proteinof HIV. Other heterologous antigens include, but are not limited to, HIVenv protein or its component parts gp120 and gp41, HIV nef protein, andthe HIV pol proteins, reverse transcriptase and protease. Still otherheterologous antigens can be those related to hepatitis C virus (HCV),including but not limited to the E1 and E2 glycoproteins, as well asnon-structural (NS) proteins, for example NS3. In addition, other viralantigens such as herpesvirus proteins may be useful. The heterologousantigens need not be limited to being of viral origin. Parasiticantigens, such as, for example, malarial antigens, are included, as arefungal antigens, bacterial antigens and tumor antigens.

As noted herein, a number of proteins expressed by tumor cells are alsoknown and are of interest as heterologous antigens which may be insertedinto the vaccine strain of the invention. These include, but are notlimited to, the bcr/abl antigen in leukemia, HPVE6 and E7 antigens ofthe oncogenic virus associated with cervical cancer, the MAGE1 and MZ2-Eantigens in or associated with melanoma, and the MVC-1 and HER-2antigens in or associated with breast cancer. Other coding sequences ofinterest include, but are not limited to, costimulatory molecules,immunoregulatory molecules, and the like.

The introduction of DNA encoding a heterologous antigen into a strain ofListeria may be accomplished, for example, by the creation of arecombinant Listeria in which DNA encoding the heterologous antigen isharbored on a vector, such as a plasmid for example, which plasmid ismaintained and expressed in the Listeria species, and in whose antigenexpression is under the control of prokaryotic promoter/regulatorysequences. Alternatively, DNA encoding the heterologous antigen may bestably integrated into the Listeria chromosome by employing, forexample, transposon mutagenesis, homologous recombination, or integrasemediated site-specific integration (as described in U.S. patentapplication Ser. No. 10/136,860, the disclosure of which is hereinincorporated by reference).

Several approaches may be employed to express the heterologous antigenin Listeria species as will be understood by one skilled in the art oncearmed with the present disclosure. In certain embodiments, genesencoding heterologous antigens are designed to either facilitatesecretion of the heterologous antigen from the bacterium or tofacilitate expression of the heterologous antigen on the Listeria cellsurface.

In certain embodiments, a fusion protein which includes the desiredheterologous antigen and a secreted or cell surface protein of Listeriais employed. Listerial proteins which are suitable components of suchfusion proteins include, but are not limited to, ActA, listeriolysin O(LLO) and phosphatidylinositol-specific phospholipase (PI-PLC). A fusionprotein may be generated by ligating the genes which encode each of thecomponents of the desired fusion protein, such that both genes are inframe with each other. Thus, expression of the ligated genes results ina protein comprising both the heterologous antigen and the Listerialprotein. Expression of the ligated genes may be placed under thetranscriptional control of a Listerial promoter/regulatory sequence suchthat expression of the gene is effected during growth and replication ofthe organism. Signal sequences for cell surface expression and/orsecretion of the fused protein may also be added to genes encodingheterologous antigens in order to effect cell surface expression and/orsecretion of the fused protein. When the heterologous antigen is usedalone (i.e., in the absence of fused Listeria sequences), it may beadvantageous to fuse thereto signal sequences for cell surfaceexpression and/or secretion of the heterologous antigen. The proceduresfor accomplishing this are well know in the art of bacteriology andmolecular biology.

The DNA encoding the heterologous antigen which is expressed is, in manyembodiments, preceded by a suitable promoter to facilitate suchexpression. The appropriate promoter/regulatory and signal sequences tobe used will depend on the type of Listerial protein desired in thefusion protein and will be readily apparent to those skilled in the artof Listeria molecular biology. For example, suitable L. monocytogenespromoter/regulatory and/or signal sequences which may be used to directexpression of a fusion protein include, but are not limited to,sequences derived from the Listeria hly gene which encodes LLO, theListeria p60 (iap) gene, and the Listeria actA gene which encodes asurface protein necessary for L. monocytogenes actin assembly. Otherpromoter sequences of interest include the plcA gene which encodesPI-PLC, the Listeria mpl gene, which encodes a metalloprotease, and theListeria inlA gene which encodes internalin, a Listeria membraneprotein. The heterologous regulatory elements such as promoters derivedfrom phage and promoters or signal sequences derived from otherbacterial species may be employed for the expression of a heterologousantigen by the Listeria species.

In certain embodiments, the mutant Listeria include a vector. The vectormay include DNA encoding a heterologous antigen. Typically, the vectoris a plasmid that is capable of replication in Listeria. The vector mayencode a heterologous antigen, wherein expression of the antigen isunder the control of eukaryotic promoter/regulatory sequences, e.g., ispresent in an expression cassette. Typical plasmids having suitablepromoters that are of interest include, but are not limited to, pCMV-βcomprising the immediate early promoter/enhancer region of humancytomegalovirus, and those which include the SV40 early promoter regionor the mouse mammary tumor virus LTR promoter region.

As such, in certain embodiments, the subject bacteria include at leastone coding sequence for heterologous polypeptide/protein, as describedabove. In many embodiments, this coding sequence is part of anexpression cassette, which provides for expression of the codingsequence in the Listeria cell for which the vector is designed. The term“expression cassette” as used herein refers to an expression module orexpression construct made up of a recombinant DNA molecule containing atleast one desired coding sequence and appropriate nucleic acid sequencesnecessary for the expression of the operably linked coding sequence in aparticular host organism, i.e., the Listeria cell for which the vectoris designed, such as the promoter/regulatory/signal sequences identifiedabove, where the expression cassette may include coding sequences fortwo or more different polypeptides, or multiple copies of the samecoding sequence, as desired. The size of the coding sequence and/orexpression cassette that includes the same may vary, but typically fallswithin the range of about 25-30 to about 6000 bp, usually from about 50to about 2000 bp. As such, the size of the encoded product may varygreatly, and a broad spectrum of different products may be encoded bythe expression cassettes present in the vectors of this embodiment.

As indicated above, the vector may include at least one coding sequence,where in certain embodiments the vectors include two or more codingsequences, where the coding sequences may encode products that actconcurrently to provide a desired results. In general, the codingsequence may encode any of a number of different products and may be ofa variety of different sizes, where the above discussion merely providesrepresentative coding sequences of interest.

c-di-AMP activity modulatory agents may also include nucleicacid/protein agents. For example, in addition to bacteria, e.g., asdescribed above, c-di-AMP activity may be modulated by providing adesired di-adenylate cyclase activity and/or c-di-adenylatephosphodiesterase activity in the target cell. As such, a target cellmay be contacted with an agent that modulates di-adenylate cyclaseactivity in the cell in a desired manner, e.g., an agent that increasesdi-adenylate cyclase activity in the cell. Examples of such agentsinclude, but are not limited to, a polypeptide comprising a DAC domain,or a nucleic acid encoding a polypeptide comprising a DAC domain asdescribed in greater detail above, e.g. a Imo2120 gene or DAC-activemutant thereof, or a DisA gene or DAC-active fragment thereof. which maybe present in a vector and/or expression cassette, at desired.Alternatively or in addition, a target cell may be contacted with anagent that modulates c-di-AMP phosphodiesterase activity in a desiredmanner, e.g., an agent that decreases di-adenylate cyclase activity inthe cell, such as a phosphodiesterase (PDE) inhibitor, a c-di-AMPphosphodiesterase-specific siRNA, etc.

In practicing methods according to embodiments of the invention, aneffective amount of the active agent, i.e., a c-di-AMP activitymodulatory agent (such as described above), is provided in the targetcell or cells. As used herein “effective amount” or “efficacious amount”means the amount of an organism or compound that, when contacted withthe cell, e.g., by being introduced into the cell in vitro, by beingadministered to a subject, etc., is sufficient to result in the desiredc-di-AMP activity modulation. The “effective amount” will vary dependingon cell and/or the organism and/or compound and or the nature of thedesired outcome and/or the disease and its severity and the age, weight,etc., of the subject to be treated. In some instances, the effectiveamount of the modulatory agent is provided in the cell by contacting thecell with the modulatory agent. Contact of the cell with the modulatoryagent may occur using any convenient protocol. The protocol may providefor in vitro or in vivo contact of the modulatory agent with the targetcell, depending on the location of the target cell. For example, wherethe target cell is an isolated cell, e.g. a cell in vitro (i.e. inculture), or a cell ex vivo (“ex vivo” being cells or organs aremodified outside of the body, where such cells or organs are typicallyreturned to a living body), and the modulatory agent is an agent thatmodulates expression of a di-adenylate cyclase or phosphodiesterase, themodulatory agent may be introduced directly into the cell under cellculture conditions permissive of viability of the target cell. Suchtechniques include, but are not necessarily limited to: viral infection,transfection, conjugation, protoplast fusion, electroporation, particlegun technology, calcium phosphate precipitation, direct microinjection,viral vector delivery, and the like. The choice of method is generallydependent on the type of cell being contacted and the nature of themodulatory agent, and the circumstances under which the transformationis taking place (e.g., in vitro, ex vivo, or in vivo). A generaldiscussion of these methods can be found in Ausubel, et al, ShortProtocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995. As anotherexample, where the target cell or cells are part of a multicellularorganism, the modulatory agent may be administered to the organism orsubject in a manner such that the agent is able to contact the targetcell(s), e.g., via an in vivo protocol. By “in vivo,” it is meant in thetarget construct is administered to a living body of an animal.

In some embodiments, the c-di-AMP activity modulatory agent is employedto modulate c-di-AMP activity in mitotic or post-mitotic cells in vitroor ex vivo, i.e., to produce modified cells that can be reintroducedinto an individual. Mitotic and post-mitotic cells of interest in theseembodiments include any eukaryotic cell, e.g. pluripotent stem cells,for example, ES cells, iPS cells, and embryonic germ cells; somaticcells, for example, hematopoietic cells, fibroblasts, neurons, musclecells, bone cells, vascular endothelial cells, gut cells, and the like,and their lineage-restricted progenitors and precursors; and neoplastic,or cancer, cells, i.e. cells demonstrating one or more propertiesassociated with cancer cells, e.g. hyperproliferation, contactinhibition, the ability to invade other tissue, etc. In certainembodiments, the eukaryotic cells are cancer cells. In certainembodiments, the eukaryotic cells are hematopoietic cells, e.g.macrophages, NK cells, etc. Cells may be from any mammalian species,e.g. murine, rodent, canine, feline, equine, bovine, ovine, primate,human, etc. Cells may be from established cell lines or they may beprimary cells, where “primary cells”, “primary cell lines”, and “primarycultures” are used interchangeably herein to refer to cells and cellscultures that have been derived from a subject and allowed to grow invitro for a limited number of passages, i.e. splittings, of the culture.For example, primary cultures are cultures that may have been passaged 0times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but notenough times go through the crisis stage. Typically, the primary celllines of the present invention are maintained for fewer than 10 passagesin vitro.

If the cells are primary cells, they may be harvest from an individualby any convenient method. For example, blood cells, e.g. leukocytes,e.g. macrophages, may be harvested by apheresis, leukocytapheresis,density gradient separation, etc., while cells from tissues such asskin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine,stomach, etc. may be harvested by biopsy. An appropriate solution may beused for dispersion or suspension of the harvested cells. Such solutionwill generally be a balanced salt solution, e.g. normal saline, PBS,Hank's balanced salt solution, etc., conveniently supplemented withfetal calf serum or other naturally occurring factors, in conjunctionwith an acceptable buffer at low concentration, generally from 5-25 mM.Convenient buffers include HEPES, phosphate buffers, lactate buffers,etc. The cells may be used immediately, or they may be stored, frozen,for long periods of time, being thawed and capable of being reused. Insuch cases, the cells will usually be frozen in 10% DMSO, 50% serum, 40%buffered medium, or some other such solution as is commonly used in theart to preserve cells at such freezing temperatures, and thawed in amanner as commonly known in the art for thawing frozen cultured cells.

As mentioned above, the c-di-AMP activity modulatory agent may beprovided to the cells as nucleic acids that encode for the c-di-AMPactivity modulatory agent, e.g. a nucleic acid that encodes for apolypeptide comprising a DAC domain to increase di-AMP activity in thecell, or a nucleic acid that encodes for a phosphodiesterase to decreasedi-AMP activity in the cell. mRNA encoding di-AMP activity modulatoryagent(s) may be provided to the cells using well-developed transfectiontechniques; see, e.g. Angel and Yanik (2010) PLoS ONE 5 (7): e11756,Beumer et al. (2008) PNAS 105 (50):19821-19826, and the commerciallyavailable TransMessenger® reagents from Qiagen, Stemfect™ RNATransfection Kit from Stemgent, and TransIT®-mRNA Transfection Kit fromMirus Bio LLC. Alternatively, nucleic acids encoding di-AMP activitymodulatory agent(s) may be provided on DNA vectors. Many vectors, e.g.plasmids, cosmids, minicircles, phage, viruses, etc., useful fortransferring nucleic acids into target cells are available. The vectorscomprising the nucleic acid(s) may be maintained episomally, e.g. asplasmids, minicircle DNAs, viruses such cytomegalovirus, adenovirus,etc., or they may be integrated into the target cell genome, throughhomologous recombination or random integration, e.g. retrovirus-derivedvectors such as MMLV, HIV-1, ALV, AAV, etc.

Vectors may be provided directly to the subject cells. In other words,the cells are contacted with vectors comprising the nucleic acidencoding the c-di-AMP activity modulatory agent(s) such that the vectorsare taken up by the cells. Methods for contacting cells with nucleicacid vectors that are plasmids, such as electroporation, calciumchloride transfection, and lipofection, are well known in the art. Forviral vector delivery, the cells are contacted with viral particlescomprising the nucleic acid encoding the c-di-AMP activity modulatoryagent(s). Retroviruses, for example, lentiviruses, are particularlysuitable to the method of the invention. Commonly used retroviralvectors are “defective”, i.e. unable to produce viral proteins requiredfor productive infection. Rather, replication of the vector requiresgrowth in a packaging cell line. To generate viral particles comprisingnucleic acids of interest, the retroviral nucleic acids comprising thenucleic acid are packaged into viral capsids by a packaging cell line.Different packaging cell lines provide a different envelope protein(ecotropic, amphotropic or xenotropic) to be incorporated into thecapsid, this envelope protein determining the specificity of the viralparticle for the cells (ecotropic for murine and rat; amphotropic formost mammalian cell types including human, dog and mouse; and xenotropicfor most mammalian cell types except murine cells). The appropriatepackaging cell line may be used to ensure that the cells are targeted bythe packaged viral particles. Methods of introducing the retroviralvectors comprising the nucleic acid encoding the reprogramming factorsinto packaging cell lines and of collecting the viral particles that aregenerated by the packaging lines are well known in the art.

Vectors used for providing the nucleic acids encoding c-di-AMP activitymodulatory agent(s) to the subject cells will typically comprisesuitable promoters for driving the expression, that is, transcriptionalactivation, of the nucleic acid of interest. In other words, the nucleicacid of interest will be operably linked to a promoter. This may includeubiquitously acting promoters, for example, the CMV-β-actin promoter, orinducible promoters, such as promoters that are active in particularcell populations or that respond to the presence of drugs such astetracycline. By transcriptional activation, it is intended thattranscription will be increased above basal levels in the target cell byat least about 10 fold, by at least about 100 fold, more usually by atleast about 1000 fold. In addition, vectors used for providing c-di-AMPactivity modulatory agent(s) to the subject cells may include nucleicacid sequences that encode for selectable markers in the target cells,so as to identify cells that have taken up the c-di-AMP activitymodulatory agent(s).

c-di-AMP activity modulatory agent(s) may also be provided to cells aspolypeptides. Such polypeptides may optionally be fused to a polypeptidedomain that increases solubility of the product. The domain may belinked to the polypeptide through a defined protease cleavage site, e.g.a TEV sequence, which is cleaved by TEV protease. The linker may alsoinclude one or more flexible sequences, e.g. from 1 to 10 glycineresidues. In some embodiments, the cleavage of the fusion protein isperformed in a buffer that maintains solubility of the product, e.g. inthe presence of from 0.5 to 2 M urea, in the presence of polypeptidesand/or polynucleotides that increase solubility, and the like. Domainsof interest include endosomolytic domains, e.g. influenza HA domain; andother polypeptides that aid in production, e.g. IF2 domain, GST domain,GRPE domain, and the like. The polypeptide may be formulated forimproved stability. For example, the peptides may be PEGylated, wherethe polyethyleneoxy group provides for enhanced lifetime in the bloodstream.

Additionally or alternatively, the c-di-AMP activity modulatory agent(s)may be fused to a polypeptide permeant domain to promote uptake by thecell. A number of permeant domains are known in the art and may be usedin the non-integrating polypeptides of the present invention, includingpeptides, peptidomimetics, and non-peptide carriers. For example, apermeant peptide may be derived from the third alpha helix of Drosophilamelanogaster transcription factor Antennapaedia, referred to aspenetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK(SEQ ID NO:016). As another example, the permeant peptide comprises theHIV-1 tat basic region amino acid sequence, which may include, forexample, amino acids 49-57 of naturally-occurring tat protein. Otherpermeant domains include poly-arginine motifs, for example, the regionof amino acids 34-56 of HIV-1 rev protein, nona-arginine, octa-arginine,and the like. (See, for example, Futaki et al. (2003) Curr Protein PeptSci. 2003 April; 4 (2): 87-96; and Wender et al. (2000) Proc. Natl.Acad. Sci. U.S.A 2000 Nov. 21; 97 (24):13003-8; published U.S. Patentapplications 20030220334; 20030083256; 20030032593; and 20030022831,herein specifically incorporated by reference for the teachings oftranslocation peptides and peptoids). The nona-arginine (R9) sequence isone of the more efficient PTDs that have been characterized (Wender etal. 2000; Uemura et al. 2002). The site at which the fusion is made maybe selected in order to optimize the biological activity, secretion orbinding characteristics of the polypeptide. The optimal site will bedetermined by routine experimentation.

The c-di-AMP activity modulatory agent(s) may be produced by eukaryoticcells or by prokaryotic cells, it may be further processed by unfolding,e.g. heat denaturation, DTT reduction, etc. and may be further refolded,using methods known in the art.

Modifications of interest that do not alter primary sequence includechemical derivatization of polypeptides, e.g., acylation, acetylation,carboxylation, amidation, etc. Also included are modifications ofglycosylation, e.g. those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps; e.g. by exposing the polypeptide to enzymes whichaffect glycosylation, such as mammalian glycosylating or deglycosylatingenzymes. Also embraced are sequences that have phosphorylated amino acidresidues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

Also included in the subject invention are c-di-AMP activity modulatoryagent(s) polypeptides that have been modified using ordinary molecularbiological techniques and synthetic chemistry so as to improve theirresistance to proteolytic degradation or to optimize solubilityproperties or to render them more suitable as a therapeutic agent.Analogs of such polypeptides include those containing residues otherthan naturally occurring L-amino acids, e.g. D-amino acids ornon-naturally occurring synthetic amino acids. D-amino acids may besubstituted for some or all of the amino acid residues.

The c-di-AMP activity modulatory agent(s) may be prepared by in vitrosynthesis, using conventional methods as known in the art. Variouscommercial synthetic apparatuses are available, for example, automatedsynthesizers by Applied Biosystems, Inc., Beckman, etc. By usingsynthesizers, naturally occurring amino acids may be substituted withunnatural amino acids. The particular sequence and the manner ofpreparation will be determined by convenience, economics, purityrequired, and the like.

If desired, various groups may be introduced into the peptide duringsynthesis or during expression, which allow for linking to othermolecules or to a surface. Thus cysteines can be used to makethioethers, histidines for linking to a metal ion complex, carboxylgroups for forming amides or esters, amino groups for forming amides,and the like.

The c-di-AMP activity modulatory agent(s) may also be isolated andpurified in accordance with conventional methods of recombinantsynthesis. A lysate may be prepared of the expression host and thelysate purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography, or other purificationtechnique. For the most part, the compositions which are used willcomprise at least 20% by weight of the desired product, more usually atleast about 75% by weight, preferably at least about 95% by weight, andfor therapeutic purposes, usually at least about 99.5% by weight, inrelation to contaminants related to the method of preparation of theproduct and its purification. Usually, the percentages will be basedupon total protein.

To modulate c-di-AMP activity, the c-di-AMP activity modulatoryagent(s)—be they polypeptides or nucleic acids that encode c-di-AMPactivity modulatory polypeptides—are provided to the cells for about 30minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours,3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12hours, 16 hours, 18 hours, 20 hours, or any other period from about 30minutes to about 24 hours, which may be repeated with a frequency ofabout every day to about every 4 days, e.g., every 1.5 days, every 2days, every 3 days, or any other frequency from about every day to aboutevery four days. The agent(s) may be provided to the subject cells oneor more times, e.g. one time, twice, three times, or more than threetimes, and the cells allowed to incubate with the agent(s) for someamount of time following each contacting event e.g. 16-24 hours, afterwhich time the media is replaced with fresh media and the cells arecultured further.

In cases in which two or more different c-di-AMP activity modulatoryagents are provided to the cell, i.e. a c-di-AMP activity modulatoryagent cocktail, the c-di-AMP activity modulatory agent(s) may beprovided simultaneously, e.g. as two polypeptides deliveredsimultaneously, as two nucleic acid vectors delivered simultaneously, oras a single nucleic acid vector comprising the coding sequences for bothfusion polypeptides. Alternatively, they may be provided consecutively,e.g. the first c-di-AMP activity modulatory agent being provided first,followed by the second c-di-AMP activity modulatory agent, etc. or viceversa.

Typically, an effective amount of c-di-AMP activity modulatory agent(s)are provided to the cells to induce a change in c-diAMP activity. Aneffective amount of c-di-AMP activity modulatory agent is the amount toinduce a 2-fold increase or more in the amount of c-di-AMP activityobserved relative to a negative control, e.g. a cell contacted with anempty vector or irrelevant polypeptide. That is to say, an effectiveamount or dose of c-di-AMP activity modulatory agent(s) will induce a2-fold increase, a 3-fold increase, a 4-fold increase or more in theamount of c-di-AMP activity observed, in some instances a 5-foldincrease, a 6-fold increase or more, sometimes a 7-fold or 8-foldincrease or more in the amount of activity observed, e.g. an increase of10-fold, 50-fold, or 100-fold or more, in some instances, an increase of200-fold, 500-fold, 700-fold, or 1000-fold or more, in the amount ofactivity observed. The amount of activity may be measured by anyconvenient method. For example, the amount of interferon produced by thecell may be assessed after contact with the c-di-AMP activity modulatoryagent(s), e.g. 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours,48 hours, 72 hours or more after contact with the c-di-AMP activitymodulatory agent(s).

Contacting the cells with the c-di-AMP activity modulatory agent(s) mayoccur in any culture media and under any culture conditions that promotethe survival of the cells. For example, cells may be suspended in anyappropriate nutrient medium that is convenient, such as Iscove'smodified DMEM or RPMI 1640, supplemented with fetal calf serum or heatinactivated goat serum (about 5-10%), L-glutamine, a thiol, particularly2-mercaptoethanol, and antibiotics, e.g. penicillin and streptomycin.The culture may contain growth factors to which the cells areresponsive. Growth factors, as defined herein, are molecules capable ofpromoting survival, growth and/or differentiation of cells, either inculture or in the intact tissue, through specific effects on atransmembrane receptor. Growth factors include polypeptides andnon-polypeptide factors.

Following the methods described above, a cell may be modified ex vivo tohave an increase in c-di-AMP activity. In some embodiments, it may bedesirous to select for the modified cell, e.g. to create an enrichedpopulation of modified cells. Any convenient modification to the cellsthat marks the cells as modified with a c-di-AMP modulatory agent may beused. For example, a selectable marker may be inserted into the genomeof the cell, so that the population of cells may be enriched for thosecomprising the genetic modification by separating the genetically markedcells from the remaining population. Separation may be by any convenientseparation technique appropriate for the selectable marker used. Forexample, if a fluorescent marker has been inserted, cells may beseparated by fluorescence activated cell sorting, whereas if a cellsurface marker has been inserted, cells may be separated from theheterogeneous population by affinity separation techniques, e.g.magnetic separation, affinity chromatography, “panning” with an affinityreagent attached to a solid matrix, or other convenient technique.Techniques providing accurate separation include fluorescence activatedcell sorters, which can have varying degrees of sophistication, such asmultiple color channels, low angle and obtuse light scattering detectingchannels, impedance channels, etc. The cells may be selected againstdead cells by employing dyes associated with dead cells (e.g. propidiumiodide). Any technique may be employed which is not unduly detrimentalto the viability of the genetically modified cells.

Cell compositions that are highly enriched for cells comprising c-di-AMPactivity modulatory agent(s) are achieved in this manner. By “highlyenriched”, it is meant that the genetically modified cells will be 70%or more, 75% or more, 80% or more, 85% or more, 90% or more of the cellcomposition, for example, about 95% or more, or 98% or more of the cellcomposition. In other words, the composition may be a substantially purecomposition of cells comprising c-di-AMP activity modulatory agent(s).

Cells comprising c-di-AMP activity modulatory agent(s) produced by themethods described herein may be used immediately. Alternatively, thecells may be frozen at liquid nitrogen temperatures and stored for longperiods of time, being thawed and capable of being reused. In suchcases, the cells will usually be frozen in 10% DMSO, 50% serum, 40%buffered medium, or some other such solution as is commonly used in theart to preserve cells at such freezing temperatures, and thawed in amanner as commonly known in the art for thawing frozen cultured cells.

The cells comprising c-di-AMP activity modulatory agent(s) may becultured in vitro under various culture conditions. The cells may beexpanded in culture, i.e. grown under conditions that promote theirproliferation. Culture medium may be liquid or semi-solid, e.g.containing agar, methylcellulose, etc. The cell population may besuspended in an appropriate nutrient medium, such as Iscove's modifiedDMEM or RPMI 1640, normally supplemented with fetal calf serum (about5-10%), L-glutamine, a thiol, particularly 2-mercaptoethanol, andantibiotics, e.g. penicillin and streptomycin. The culture may containgrowth factors to which the regulatory T cells are responsive. Growthfactors, as defined herein, are molecules capable of promoting survival,growth and/or differentiation of cells, either in culture or in theintact tissue, through specific effects on a transmembrane receptor.Growth factors include polypeptides and non-polypeptide factors.

Cells that have been modified with c-di-AMP activity modulatory agent(s)may be transplanted to a subject for purposes such as gene therapy, e.g.to treat a disease or as an antiviral, antipathogenic, or anticancertherapeutic, for the production of genetically modified organisms inagriculture, or for biological research. The subject may be a neonate, ajuvenile, or an adult. Of particular interest are mammalian subjects.Mammalian species that may be treated with the present methods includecanines and felines; equines; bovines; ovines; etc. and primates,particularly humans. Animal models, particularly small mammals, e.g.murine, lagomorpha, etc. may be used for experimental investigations.

Cells may be provided to the subject alone or with a suitable substrateor matrix, e.g. to support their growth and/or organization in thetissue to which they are being transplanted. Usually, at least 1×10³cells will be administered, for example 5×10³ cells, 1×10⁴ cells, 5×10⁴cells, 1×10⁵ cells, 1×10⁶ cells or more. The cells may be introduced tothe subject via any of the following routes: parenteral, subcutaneous,intravenous, intracranial, intraspinal, intraocular, or into spinalfluid. The cells may be introduced by injection, catheter, or the like.Examples of methods for local delivery, that is, delivery to the site ofinjury, include, e.g. through an Ommaya reservoir, e.g. for intrathecaldelivery (see e.g. U.S. Pat. Nos. 5,222,982 and 5,385,582, incorporatedherein by reference); by bolus injection, e.g. by a syringe, e.g. into ajoint; by continuous infusion, e.g. by cannulation, e.g. with convection(see e.g. US Application No. 20070254842, incorporated here byreference); or by implanting a device upon which the cells have beenreversably affixed (see e.g. US Application Nos. 20080081064 and20090196903, incorporated herein by reference).

The number of administrations of treatment to a subject may vary.Introducing the genetically modified cells into the subject may be aone-time event; but in certain situations, such treatment may elicitimprovement for a limited period of time and require an on-going seriesof repeated treatments. In other situations, multiple administrations ofthe genetically modified cells may be required before an effect isobserved. The exact protocols depend upon the disease or condition, thestage of the disease and parameters of the individual subject beingtreated.

In other aspects of the invention, the c-di-AMP activity modulatoryagent(s) are employed to modify cellular c-di-AMP activity in vivo. Inthese in vivo embodiments, the c-di-AMP activity modulatory agent(s) areadministered directly to the individual. c-di-AMP activity modulatoryagent(s) may be administered by any of a number of well-known methods inthe art for the administration of peptides, small molecules and nucleicacids to a subject. The c-di-AMP activity modulatory agent(s) can beincorporated into a variety of formulations. More particularly, thec-di-AMP activity modulatory agent(s) of the present invention can beformulated into pharmaceutical compositions by combination withappropriate pharmaceutically acceptable carriers or diluents.

In the subject methods, the active agent(s) may be administered to thetargeted cells using any convenient administration protocol capable ofresulting in the desired activity. Thus, the agent can be incorporatedinto a variety of formulations, e.g., pharmaceutically acceptablevehicles, for therapeutic administration. “Pharmaceutically acceptablevehicles” may be vehicles approved by a regulatory agency of the Federalor a state government or listed in the U.S. Pharmacopeia or othergenerally recognized pharmacopeia for use in mammals, such as humans.The term “vehicle” refers to a diluent, adjuvant, excipient, or carrierwith which a compound of the invention is formulated for administrationto a mammal. Such pharmaceutical vehicles can be lipids, e.g. liposomes,e.g. liposome dendrimers; liquids, such as water and oils, includingthose of petroleum, animal, vegetable or synthetic origin, such aspeanut oil, soybean oil, mineral oil, sesame oil and the like, saline;gum acacia, gelatin, starch paste, talc, keratin, colloidal silica,urea, and the like. In addition, auxiliary, stabilizing, thickening,lubricating and coloring agents may be used. More particularly, theagents of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments (e.g., skin creams), solutions,suppositories, injections, inhalants and aerosols. As such,administration of the agents can be achieved in various ways, includingoral, buccal, rectal, parenteral, intraperitoneal, intradermal,transdermal, intracheal, etc., administration. The active agent may besystemic after administration or may be localized by the use of regionaladministration, intramural administration, or use of an implant thatacts to retain the active dose at the site of implantation. The activeagent may be formulated for immediate activity or it may be formulatedfor sustained release.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

Typically, an effective amount of c-di-AMP activity modulatory agent(s)are provided. As discussed above with regard to ex vivo methods, aneffective amount or effective dose of a c-di-AMP activity modulatoryagent in vivo is the amount to induce a 2 fold increase or more in theamount of c-di-AMP activity in the cell relative to a negative control,e.g. a cell contacted with an empty vector, irrelevant polypeptide, etc.The amount of activity may be measured by any convenient method, e.g. asdescribed herein and known in the art. The calculation of the effectiveamount or effective dose of a c-di-AMP activity modulatory agent to beadministered is within the skill of one of ordinary skill in the art,and will be routine to those persons skilled in the art. Needless tosay, the final amount to be administered will be dependent upon theroute of administration and upon the nature of the disorder or conditionthat is to be treated.

The effective amount given to a particular patient will depend on avariety of factors, several of which will differ from patient topatient. A competent clinician will be able to determine an effectiveamount of a therapeutic agent to administer to a patient to halt orreverse the progression the disease condition as required. UtilizingLD₅₀ animal data, and other information available for the agent, aclinician can determine the maximum safe dose for an individual,depending on the route of administration. For instance, an intravenouslyadministered dose may be more than an intrathecally administered dose,given the greater body of fluid into which the therapeutic compositionis being administered. Similarly, compositions which are rapidly clearedfrom the body may be administered at higher doses, or in repeated doses,in order to maintain a therapeutic concentration. Utilizing ordinaryskill, the competent clinician will be able to optimize the dosage of aparticular therapeutic in the course of routine clinical trials.

For inclusion in a medicament, the c-di-AMP activity modulatory agent(s)may be obtained from a suitable commercial source. As a generalproposition, the total pharmaceutically effective amount of the c-di-AMPactivity modulatory agent(s) administered parenterally per dose will bein a range that can be measured by a dose response curve.

c-di-AMP activity modulatory agent-based therapies, i.e. preparations ofc-di-AMP activity modulatory agent(s) to be used for therapeuticadministration, must be sterile. Sterility is readily accomplished byfiltration through sterile filtration membranes (e.g., 0.2 μmmembranes). Therapeutic compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle. The c-di-AMP activity modulatory agent-based therapiesmay be stored in unit or multi-dose containers, for example, sealedampules or vials, as an aqueous solution or as a lyophilized formulationfor reconstitution. As an example of a lyophilized formulation, 10-mLvials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous solutionof compound, and the resulting mixture is lyophilized. The infusionsolution is prepared by reconstituting the lyophilized compound usingbacteriostatic Water-for-Injection.

Pharmaceutical compositions can include, depending on the formulationdesired, pharmaceutically-acceptable, non-toxic carriers of diluents,which are defined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, buffered water, physiologicalsaline, PBS, Ringer's solution, dextrose solution, and Hank's solution.In addition, the pharmaceutical composition or formulation can includeother carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenicstabilizers, excipients and the like. The compositions can also includeadditional substances to approximate physiological conditions, such aspH adjusting and buffering agents, toxicity adjusting agents, wettingagents and detergents.

The composition can also include any of a variety of stabilizing agents,such as an antioxidant for example. When the pharmaceutical compositionincludes a polypeptide, the polypeptide can be complexed with variouswell-known compounds that enhance the in vivo stability of thepolypeptide, or otherwise enhance its pharmacological properties (e.g.,increase the half-life of the polypeptide, reduce its toxicity, enhancesolubility or uptake). Examples of such modifications or complexingagents include sulfate, gluconate, citrate and phosphate. The nucleicacids or polypeptides of a composition can also be complexed withmolecules that enhance their in vivo attributes. Such molecules include,for example, carbohydrates, polyamines, amino acids, other peptides,ions (e.g., sodium, potassium, calcium, magnesium, manganese), andlipids.

Further guidance regarding formulations that are suitable for varioustypes of administration can be found in Remington's PharmaceuticalSciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985).For a brief review of methods for drug delivery, see, Langer, Science249:1527-1533 (1990).

The pharmaceutical compositions can be administered for prophylacticand/or therapeutic treatments. Toxicity and therapeutic efficacy of theactive ingredient can be determined according to standard pharmaceuticalprocedures in cell cultures and/or experimental animals, including, forexample, determining the LD50 (the dose lethal to 50% of the population)and the ED50 (the dose therapeutically effective in 50% of thepopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index and it can be expressed as the ratio LD50/ED50.Therapies that exhibit large therapeutic indices are preferred.

The data obtained from cell culture and/or animal studies can be used informulating a range of dosages for humans. The dosage of the activeingredient typically lines within a range of circulating concentrationsthat include the ED50 with low toxicity. The dosage can vary within thisrange depending upon the dosage form employed and the route ofadministration utilized.

The components used to formulate the pharmaceutical compositions arepreferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents, particularly any endotoxins, which may bepresent during the synthesis or purification process. Compositions forparental administration are also sterile, substantially isotonic andmade under GMP conditions.

The effective amount of a therapeutic composition to be given to aparticular patient will depend on a variety of factors, several of whichwill differ from patient to patient. A competent clinician will be ableto determine an effective amount of a therapeutic agent to administer toa patient to halt or reverse the progression the disease condition asrequired. Utilizing LD50 animal data, and other information availablefor the agent, a clinician can determine the maximum safe dose for anindividual, depending on the route of administration. For instance, anintravenously administered dose may be more than an intrathecallyadministered dose, given the greater body of fluid into which thetherapeutic composition is being administered. Similarly, compositionswhich are rapidly cleared from the body may be administered at higherdoses, or in repeated doses, in order to maintain a therapeuticconcentration. Utilizing ordinary skill, the competent clinician will beable to optimize the dosage of a particular therapeutic in the course ofroutine clinical trials.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Where the agent is a polypeptide, polynucleotide, analog or mimeticthereof, it may be introduced into tissues or host cells by any numberof routes, including viral infection, microinjection, or fusion ofvesicles. Jet injection may also be used for intramuscularadministration, as described by Furth et al. (1992), Anal Biochem205:365-368. The DNA may be coated onto gold microparticles, anddelivered intradermally by a particle bombardment device, or “gene gun”as described in the literature (see, for example, Tang et al. (1992),Nature 356:152-154), where gold microprojectiles are coated with theDNA, then bombarded into skin cells. For nucleic acid therapeuticagents, a number of different delivery vehicles find use, includingviral and non-viral vector systems, as are known in the art.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the nature of the deliveryvehicle, and the like. Preferred dosages for a given compound arereadily determinable by those of skill in the art by a variety of means.

As reviewed above, the subject methods result in the modulation ofc-di-AMP activity inside a cell, where the target cell(s) may be invitro or in vivo. In certain embodiments, the subject methods result inreduction in toxicity of a target gene, e.g., via a reduction inaggregation of a protein encoded thereby, in a target cell(s). Incertain embodiments, the methods result in enhancement in function of aprotein encoded by a target gene.

The above methods find use in a variety of different applications.Certain applications are now reviewed in the following Utility section.

Utility

The methods and compositions of the invention find use in a variety ofapplications, where such applications include modulation of interferon-βproduction in a subject is desired. Specific applications of interestinclude those in which a subject is treated for a disease condition. Insome embodiments, subjects suitable for treatment with a method of thepresent invention include individuals having a cellular proliferativedisease, such as a neoplastic disease (e.g., cancer). Cellularproliferative disease is characterized by the undesired propagation ofcells, including, but not limited to, neoplastic disease conditions,e.g., cancer. Examples of cellular proliferative disease include, butnot limited to, abnormal stimulation of endothelial cells (e.g.,atherosclerosis), solid tumors and tumor metastasis, benign tumors, forexample, hemangiomas, acoustic neuromas, neurofibromas, trachomas, andpyogenic granulomas, vascular malfunctions, abnormal wound healing,inflammatory and immune disorders, Bechet's disease, gout or goutyarthritis, abnormal angiogenesis accompanying, for example, rheumatoidarthritis, psoriasis, diabetic retinopathy, other ocular angiogenicdiseases such as retinopathy of prematurity (retrolental fibroplastic),macular degeneration, corneal graft rejection, neurovascular glaucomaand Oster Webber syndrome, psoriasis, restenosis, fungal, parasitic andviral infections such cytomegaloviral infections. Subjects to be treatedaccording to the methods of the invention include any individual havingany of the above-mentioned disorders.

In other embodiments, subjects suitable for treatment with a method ofthe present invention include individuals who have been clinicallydiagnosed as infected with a hepatitis virus (e.g., HAV, HBV, HCV,delta, etc.), particularly HCV, are suitable for treatment with themethods of the instant invention. Individuals who are infected with HCVare identified as having HCV RNA in their blood, and/or having anti-HCVantibody in their serum. Such individuals include naïve individuals(e.g., individuals not previously treated for HCV, particularly thosewho have not previously received IFN-α-based or ribavirin-based therapy)and individuals who have failed prior treatment for HCV.

In other embodiments, subjects suitable for treatment with a method ofthe present invention include individuals having multiple sclerosis.Multiple sclerosis refers to an autoimmune neurodegenerative disease,which is marked by inflammation within the central nervous system withlymphocyte attack against myelin produced by oligodendrocytes, plaqueformation and demyelization with destruction of the myelin sheath ofaxons in the brain and spinal cord, leading to significant neurologicaldisability over time. Typically, at onset an otherwise healthy personpresents with the acute or sub acute onset of neurologicalsymptomatology (attack) manifested by unilateral loss of vision,vertigo, ataxia, dyscoordination, gait difficulties, sensory impairmentcharacterized by paresthesia, dysesthesia, sensory loss, urinarydisturbances until incontinence, diplopia, dysarthria or various degreesof motor weakness until paralysis. The symptoms are usually painless,remain for several days to a few weeks, and then partially or completelyresolve. After a period of remission, a second attack will occur. Duringthis period after the first attack, the patient is defined to sufferfrom probable MS. Probable MS patients may remain undiagnosed for years.When the second attack occurs the diagnosis of clinically definite MS(CDMS) is made (Poser criteria 1983; C. M. Poser et al., Ann. Neurol.1983; 13, 227).

The terms “subject” and “patient” mean a member or members of anymammalian or non-mammalian species that may have a need for thepharmaceutical methods, compositions and treatments described herein.Subjects and patients thus include, without limitation, primate(including humans), canine, feline, ungulate (e.g., equine, bovine,swine (e.g., pig)), avian, and other subjects. Humans and non-humananimals having commercial importance (e.g., livestock and domesticatedanimals) are of particular interest.

“Mammal” means a member or members of any mammalian species, andincludes, by way of example, canines; felines; equines; bovines; ovines;rodentia, etc. and primates, particularly humans. Non-human animalmodels, particularly mammals, e.g. primate, murine, lagomorpha, etc. maybe used for experimental investigations.

“Treating” or “treatment” of a condition or disease includes: (1)preventing at least one symptom of the conditions, i.e., causing aclinical symptom to not significantly develop in a mammal that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease, (2) inhibiting the disease, i.e.,arresting or reducing the development of the disease or its symptoms, or(3) relieving the disease, i.e., causing regression of the disease orits clinical symptoms. As used herein, the term “treating” is thus usedto refer to both prevention of disease, and treatment of pre-existingconditions. For example, where the mutant bacteria is administered, theprevention of cellular proliferation can be accomplished byadministration of the subject compounds prior to development of overtdisease, e.g. to prevent the regrowth of tumors, prevent metastaticgrowth, etc. Alternatively the compounds are used to treat ongoingdisease, by stabilizing or improving the clinical symptoms of thepatient.

Combination Therapy

For use in the subject methods, the c-di-AMP modulatory agents, such asthe subject mutant Listeria described above, may be administered incombination with other pharmaceutically active agents, including otheragents that treat the underlying condition or a symptom of thecondition. In addition, the mutant Listeria may be used to provide anincrease in the effectiveness of another chemical, such as apharmaceutical, that is necessary to produce the desired biologicaleffect.

“In combination with” as used herein refers to uses where, for example,the first compound is administered during the entire course ofadministration of the second compound; where the first compound isadministered for a period of time that is overlapping with theadministration of the second compound, e.g. where administration of thefirst compound begins before the administration of the second compoundand the administration of the first compound ends before theadministration of the second compound ends; where the administration ofthe second compound begins before the administration of the firstcompound and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe first compound begins before administration of the second compoundbegins and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe second compound begins before administration of the first compoundbegins and the administration of the first compound ends before theadministration of the second compound ends. As such, “in combination”can also refer to regimen involving administration of two or morecompounds. “In combination with” as used herein also refers toadministration of two or more compounds which may be administered in thesame or different formulations, by the same of different routes, and inthe same or different dosage form type.

Examples of other agents for use in combination therapy of neoplasticdisease include, but are not limited to, thalidomide, marimastat, COL-3,BMS-275291, squalamine, 2-ME, SU6668, neovastat, Medi-522, EMD121974,CAI, celecoxib, interleukin-12, IM862, TNP470, avastin, gleevec,herceptin, and mixtures thereof. Examples of chemotherapeutic agents foruse in combination therapy include, but are not limited to,daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosinearabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphor-amide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES).

Other antiviral agents can also be delivered in the treatment methods ofthe invention. For example, compounds that inhibit inosine monophosphatedehydrogenase (IMPDH) may have the potential to exert direct anti viralactivity, and such compounds can be administered in combination with themutant Listeria, as described herein. Drugs that are effectiveinhibitors of hepatitis C NS3 protease may be administered incombination with the mutant Listeria, as described herein. Hepatitis CNS3 protease inhibitors inhibit viral replication. Other agents such asinhibitors of HCV NS3 helicase are also attractive drugs forcombinational therapy, and are contemplated for use in combinationtherapies described herein. Ribozymes such as Heptazyme™ andphosphorothioate oligonucleotides which are complementary to HCV proteinsequences and which inhibit the expression of viral core proteins arealso suitable for use in combination therapies described herein.Examples of other agents for use in combination therapy of multiplesclerosis include, but are not limited to; glatiramer; corticosteroids;muscle relaxants, such as Tizanidine (Zanaflex) and baclofen (Lioresal);medications to reduce fatigue, such as amantadine (Symmetrel) ormodafinil (Provigil); and other medications that may also be used fordepression, pain and bladder or bowel control problems that can beassociated with MS.

In the context of a combination therapy, combination therapy compoundsmay be administered by the same route of administration (e.g.intrapulmonary, oral, enteral, etc.) that the mutant Listeria areadministered. In the alternative, the compounds for use in combinationtherapy with the mutant Listeria may be administered by a differentroute of administration.

Adjuvant Compositions

The subject mutant bacterial strains, e.g., as described above, alsofind use as immunopotentiating agents, i.e., as adjuvants. In suchapplications, the subject attenuated bacteria may be administered inconjunction with an immunogen, e.g., a tumor antigen, modified tumorcell, etc., according to methods known in the art where live bacterialstrains are employed as adjuvants. See, e.g., Berd et al., Vaccine 2001Mar. 21; 19 (17-19):2565-70.

In some embodiments, the mutant bacterial strains are employed asadjuvants by chemically coupling them to a sensitizing antigen. Thesensitizing antigen can be any antigen of interest, where representativeantigens of interest include, but are not limited to: viral agents,e.g., Herpes simplex virus; malaria parasite; bacteria, e.g.,staphylococcus aureus bacteria, diphtheria toxoid, tetanus toxoid,shistosomula; tumor cells, e.g. CAD2 mammary adenocarcinomia tumorcells, and hormones such as thyroxine T4, triiiodothyronine T3, andcortisol. The coupling of the sensitizing antigen to theimmunopotentiating agent can be accomplished by means of variouschemical agents having two reactive sites such as, for example,bisdiazobenzidine, glutaraldehyde, di-iodoacetate, and diisocyanates,e.g., m-xylenediisocyanate and toluene-2,4-diisocyanate. Use of Listeriaspp. as adjuvants is further described in U.S. Pat. No. 4,816,253.

Vaccines

The subject bacteria, e.g., as described above, also find use asvaccines. The vaccines of the present invention are administered to avertebrate by contacting the vertebrate with a sublethal dose of anattenuated mutant Listeria vaccine, where contact typically includesadministering the vaccine to the host. In many embodiments, theattenuated bacteria are provided in a pharmaceutically acceptableformulation. Administration can be oral, parenteral, intranasal,intramuscular, intradermal, intraperitoneal, intravascular,subcutaneous, direct vaccination of lymph nodes, administration bycatheter or any one or more of a variety of well-known administrationroutes. In farm animals, for example, the vaccine may be administeredorally by incorporation of the vaccine in feed or liquid (such aswater). It may be supplied as a lyophilized powder, as a frozenformulation or as a component of a capsule, or any other convenient,pharmaceutically acceptable formulation that preserves the antigenicityof the vaccine. Any one of a number of well known pharmaceuticallyacceptable diluents or excipients may be employed in the vaccines of theinvention. Suitable diluents include, for example, sterile, distilledwater, saline, phosphate buffered solution, and the like. The amount ofthe diluent may vary widely, as those skilled in the art will recognize.Suitable excipients are also well known to those skilled in the art andmay be selected, for example, from A. Wade and P. J. Weller, eds.,Handbook of Pharmaceutical Excipients (1994) The Pharmaceutical Press:London. The dosage administered may be dependent upon the age, healthand weight of the patient, the type of patient, and the existence ofconcurrent treatment, if any. The vaccines can be employed in dosageforms such as capsules, liquid solutions, suspensions, or elixirs, fororal administration, or sterile liquid for formulations such assolutions or suspensions for parenteral, intranasal intramuscular, orintravascular use. In accordance with the invention, the vaccine may beemployed, in combination with a pharmaceutically acceptable diluent, asa vaccine composition, useful in immunizing a patient against infectionfrom a selected organism or virus or with respect to a tumor, etc.Immunizing a patient means providing the patient with at least somedegree of therapeutic or prophylactic immunity against selectedpathogens, cancerous cells, etc.

The subject vaccines find use in methods for eliciting or boosting acellular immune response, e.g., a helper T cell or a cytotoxic T-cellresponse to a selected agent, e.g., pathogenic organism, tumor, etc., ina vertebrate, where such methods include administering an effectiveamount of the Listeria vaccine. The subject vaccines find use in methodsfor eliciting in a vertebrate an innate immune response that augmentsthe antigen-specific immune response. Furthermore, the vaccines of thepresent invention may be used for treatment post-exposure or postdiagnosis. In general, the use of vaccines for post-exposure treatmentwould be recognized by one skilled in the art, for example, in thetreatment of rabies and tetanus. The same vaccine of the presentinvention may be used, for example, both for immunization and to boostimmunity after exposure. Alternatively, a different vaccine of thepresent invention may be used for post-exposure treatment, for example,such as one that is specific for antigens expressed in later stages ofexposure. As such, the subject vaccines prepared with the subjectvectors find use as both prophylactic and therapeutic vaccines to induceimmune responses that are specific for antigens that are relevant tovarious disease conditions.

The patient may be any human and non-human animal susceptible toinfection with the selected organism. The subject vaccines will findparticular use with vertebrates such as man, and with domestic animals.Domestic animals include domestic fowl, bovine, porcine, ovine, equine,caprine, Leporidate (such as rabbits), or other animal which may be heldin captivity.

In certain instances, the patient is one that has been predetermined,e.g., diagnosed, to be in need of type-I interferon productionmodulation. In some instances, the methods may include diagnosing thepatient to be in need of type-I interferon production.

In general, the subject vaccines find use in vaccination applications asdescribed U.S. Pat. Nos. 5,830,702 and 6,051,237, as well as PCTpublication no WO 99/25376.

Kits

Kits with unit doses of the subject c-di-AMP activity modulatory agents,e.g., mutant Listeria, e.g., in oral or injectable doses, are provided.In the subject kits, the one or more components are present in the sameor different containers, as may be convenient or desirable.

In addition to the containers containing the unit doses will beinstructions describing the use and attendant benefits of the mutantListeria in treating a pathological condition of interest. Instructionsmay be provided in a variety of different formats. In certainembodiments, the instructions may include complete protocols forpracticing the subject methods or means for obtaining the same (e.g., awebsite URL directing the user to a webpage which provides theinstructions), where these instructions may be printed on a substrate,where substrate may be one or more of: a package insert, the packaging,reagent containers and the like.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

I. Overview

Listeria monocytogenes (Lm) is a gram-positive food-born pathogen.Eradication of Lm infection relies on an adaptive cell-mediated immuneresponse. This has led to the use of Lm as a vaccine platform,engineered to generate pathogen and tumor specific antigens that induceimmunity toward a desired target. Such immunotherapies show particularpromise for treatment of infectious and malignant disease. The successof genetically modified Lm as a vaccine is due to its ability toactivate an innate immune response upon cytosolic entry, which iscrucial to obtain optimal immunogenicity. Activation of a distinct hostresponse upon cytosolic entry has led to the hypothesis of a cytosollicsurveillance pathway (CSP), a branch of the innate immune system able tomonitor the infiltration of the cytosol by microbes.¹ Here we identifycyclic di-adenosine monophosphate (c-di-AMP) as a CSP activating smallmolecule derived from Lm.

Here, we establish cytosolic sensing of c-di-AMP and show that thisligand contributes to type I interferon production during L.monocytogenes infection of macrophages. Given the importance of type-Iinterferon immunotherapy for malignant and chronic viral treatments andthe promise of Lm as a vaccine platform, c-di-AMP, synthetic analogs,and Lm strains with variable production/secretion of c-di-AMP find useas adjuvant strains for vaccine development and IFN-βimmunotherapeutics.

One major contribution of this work is the identification of type-Iinterferon induction by c-di-AMP. IFN-β immunotherapy shows promise incancer drug regimens because of its anti-proliferative, anti-angiogenic,and immunomodulating effects on many human cancers. (Borden, E. C., JInterferon Cytokine Res 2005, 25, 511-527) However, IFN-β is limited byits short serum half-life. A small molecule,5,6-dimethylxanthenone-4-acetic acid (DMXAA), currently in phase IIIclinical trials for cancer treatment, is therapeutically active due toits ability to stimulate IFN-β production. Given the small, drug-likenature of c-di-AMP, this molecule or synthetic analogs thereof areuseful as IFN-β inducing therapeutics, much like DMXAA. Furthermore, Lmstrains that have increased levels of c-di-AMP secretion also find useas type-I IFN inducing therapeutics.

Another contribution of this work is the establishment of cytosolicsensing of the c-di-AMP. One significant advantage of using Lm as adelivery platform for c-di-AMP is that it allows the molecule to gainaccess to the cytosol, where the host detects it. Given that c-di-AMP isnot cell permeable and does not induce inflammation when added to theoutside of cells, this method will offer significant advantage overusing the purified molecule as an adjuvant or IFN inducing agent, wherecytosolic delivery is difficult to attain.

Two families of proteins have recently been described, the di-adenylatecyclase domain (DAC, previously DUF147) that catalyzes the formation ofc-di-AMP, and the DHH/DHHA1 domain that is a c-di-AMP phosphodiesterase(Rao, F. et al. J Biol Chem 2009; Witte, G et al. Mol Cell 2008, 30,167-178). Listeria has one known di-adenylate cyclase, Imo2120, and oneknown c-di-AMP phosphodiesterase, Imo0052. Deletion or overexpression ofeach of these proteins or heterologous proteins with similar activity inLm results in altered levels of c-di-AMP production and IFN productionduring host infection. These proteins can either be expressed in thebacterium to alter secretion levels of c-di-AMP or secreted to alter theextracellular concentration of the nucleotide. For instance, secretionof the protein Imo2120 into the cytosol of the host where ample levelsof ATP, the substrate for the DAC, are present leads to the generationof c-di-AMP directly in the cytosol to enhance IFN production from theinfected cell. Conversely, secretion of the c-di-AMP specificphosphodiesterase destroys secreted c-di-AMP and results in lower levelsof IFN produced during infection. Such strains that generate alteredlevels of c-di-AMP may be rationally designed as vaccine platforms andimmunotherapeutics for treatment of chronic and malignant disease.

II. Cyclic di-AMP Secreted by Listeria monocytogenes MDRs Activates aHost Cytosolic Surveillance Pathway A. Introduction

Intracellular pathogens, such as Listeria monocytogenes, are detected inthe cytosol of host immune cells, although the activating ligand(s) arenot known. Induction of this host response is often dependent onmicrobial secretion systems, and in L. monocytogenes, it is dependent onmultidrug efflux pumps (MDRs) of the MFS family. Using L. monocytogenesmutants that over-express MDRs, we identified cyclic diadenosinemonophosphate (c-di-AMP) as a secreted molecule able to activate thecytosolic host response. Over-expression of the di-adenylate cyclase,dacA (Imo2120), resulted in elevated levels of the host response duringinfection. These studies identify a small molecule (c-di-AMP), predictedto be present in a wide variety of bacteria and archea, that triggers acytosolic pathway of innate immunity, and are consistent with thisnucleotide being secreted by bacterial MDRs.

Intracellular pathogens, such as Listeria monocytogenes, are detected inthe cytosol of host immune cells leading to transcription of type Iinterferon and co-regulated genes. Although the activating ligand(s) areunknown, induction of this host response is often dependent on microbialsecretion systems, and in L. monocytogenes, it is dependent on multidrugefflux pumps (MDRs) of the MFS family. Using L. monocytogenes mutantsthat over-express MDRs, we identified cyclic diadenosine monophosphate(c-di-AMP) as a secreted molecule able to activate the cytosolic hostresponse. Over-expression of the di-adenylate cyclase, dacA (Imo2120),resulted in elevated levels of the host response during infection. Thesestudies identify a small signalling molecule (c-di-AMP), predicted to bepresent in a wide variety of bacteria and archea, that triggers acytosolic pathway of innate immunity.

B. Results

The mammalian innate immune system is comprised of receptors thatcollectively serve as a pathogen sensor to monitor the extracellular,vacuolar and cytosolic cellular compartments.¹ Recognition of microbeswithin these distinct compartments leads to cellular responses that arecommensurate with the microbial threat. While both pathogenic andnon-pathogenic microbes interact with extracellular and vacuolarcompartments, infectious disease agents often mediate their pathogenesisby delivery of virulence factors directly into the host cell cytosoliccompartment. Thus, an attractive hypothesis has emerged that the innateimmune system distinguishes between pathogenic and non-pathogenicmicrobes by monitoring the sanctity of the cytosol.^(2, 3)

Several distinct pathways of innate immunity are present in the hostcell cytosol. One, termed the cytosolic surveillance pathway (CSP),detects bacterial, viral and protozoan pathogens, leading to theactivation of IRF3 and NF-κB and induction of IFN-β and co-regulatedgenes.⁴ Some ligands that activate this pathway are known, for example,viral nucleic acids. However, the ligands and host receptors that leadto IFN-β by non-viral microbes, including L. monocytogenes, M.tuberculosis, F. tularensis, L. pneumophila, B. abortis, and T. cruzi,among others, remain unknown.⁴⁻⁹

In a previous study, we used a genetic screen to search for microbialfactors that affect this host innate immune pathway.¹⁰ We found thatexpression of L. monocytogenes multidrug efflux pumps (MDRs) of themajor facilitator superfamily controlled the capacity of cytosolicbacteria to induce host expression of IFN-β. Ectopic expression ofmultiple MDRs led to enhanced IFN-β production, while one, MdrM,controlled the majority of the response to wild-type bacteria.

Given that MDRs are known to transport small molecules (<1000 Da), wehypothesized that L. monocytogenes secretes a bioactive small moleculethat is recognized within the host cytosol. However, over-expression ofbacterial MDRs may compromise membrane integrity and increase deliveryof microbial ligands non-specifically. Non-specific delivery ofbacterial ligand(s) during infection would, therefore, be independent oftransport activity of MDRs but dependent on transporter expression. Toaddress these two possibilities, L. monocytogenes strains thatover-express transport inactive mutants of MdrM were generated.Hexa-histidine tagged, site-directed mutants (R109A and G154C) and WTMdrM under control of IPTG induction were introduced into the mdrM-L.monocytogenes strain. These two specific residues are conserved in MFSfamily MDRs and are crucial to transport activity.¹¹⁻¹³ Comparablemembrane incorporation of wild-type and mutant variants of MdrM andsimilar intracellular growth of each strain was observed by western-blotof membrane fractions (FIG. 5 for representative blot) and infection ofbone marrow derived macrophages, respectively (FIG. 1 a-b). Only the WT,transport-competent expressing strain activated the CSP (FIG. 1 c).These results are consistent with the need for transport activity ofMDRs to induce IFN-β expression during infection and secretion of anactive small molecule by these transporters.

To identify the bioactive ligand(s) secreted by L. monocytogenes MDRs,we performed solid phase extraction (SPE) of the culture supernatantfrom an MdrM over-expressing L. monocytogenes strain (marR-, DP-L5445)that exhibits an IFN-β hyper-activating phenotype. Delivery of thefraction to the macrophage cytosol using reversible digitoninpermeabilization¹⁴ resulted in a dose-dependent increase in type-I IFN(FIG. 2 a). Addition of this fraction in the absence of digitoninresulted in no IFN production, consistent with cytosolic detection ofthe active ligand.

Previous characterization of L. monocytogenes strains that exhibitvariable levels of MDR expression demonstrated that IFN-β productioncorrelates with increases in transporter levels.¹⁰ Here, supernatantsfrom four L. monocytogenes strains, mdrM-, WT, marR-, and tetR::Tn917,that exhibit increasing levels of MDR expression, respectively, weretested for activity. Comparable to infection assays, MDR expressioncorrelated with IFN inducing activity of the culture supernatants (FIG.2 b), where the tetR::Tn917 strain exhibited significantly higheractivity than any other strain, while the mdrM-strain lacked detectableactivity above background.

Fractionation of the active samples obtained from each MDR strain wasperformed using reversed-phase high performance liquid chromatography(RP-HPLC). The active component of each supernatant eluted as a singlepeak from the column with similar retention time (FIG. 2 c), consistentwith each containing the same active ligand. Furthermore, the magnitudeof the active peak correlated with MDR expression. The sample with thehighest activity exhibited a significant absorbance at 260 nm (FIG. 6a). Incubation with anion but not cation exchange resin removed theactive molecule from solution (FIG. 6 b), although treatment of theactive sample was resistant to DNAse (FIG. 6 c). These results wereconsistent with a non-DNA, nucleic acid as the active component.

To identify the IFN-β inducing metabolite contained in the fractions,samples were analyzed by high-resolution mass spectrometry. A single ion(m/z=659.11, z=1) was identified as exclusively present in the activefractions and absent in the inactive samples (FIG. 7 a). The parent ionmass were consistent with cyclic di-adenosine monophosphate (c-di-AMP,FIG. 7 b). Collision induced dissociation was performed to furthercharacterize the identified ion (FIG. 3 a). Comparison of thefragmentation pattern with synthetic standard (BioLog Life ScienceInstitute, Denmark) confirmed the assignment of the ion (FIGS. 3 b-c).Quantification of c-di-AMP in these samples revealed that the mdrM-, WT,and marR-strains had 23%, 34%, and 35% as much c-di-AMP in the culturesupernatants relative to tetR::Tn917 (53 nM), respectively. Theseresults established that IFN-β inducing activity of L. monocytogenessupernatants correlates linearly with c-di-AMP concentration (FIG. 8).

Next, we tested the ability of commercially available c-di-AMP to induceIFN-β in macrophages. Similar to the active sample, c-di-AMP exhibits adose-dependent response when delivered to the cytosol of murine BMMs(FIG. 3 d). Treatment of the purified active fraction and the commercialstandard with snake venom phosphodiesterase (SVPD) abolished theactivity of each sample. The host pathway responsible for cytosolicdetection of L. monocytogenes is dependent on IRF3 and STING butindependent of MyD88/Trif and MAVS.^(10, 15) Detection of c-di-AMPrequires a parallel host-signaling pathway, consistent with c-di-AMP asthe relevant ligand of L. monocytogenes (FIG. 9). The above resultsdemonstrate that the intracellular pathogen L. monocytogenes generatesthe novel nucleotide, c-di-AMP, which induces the host cytosolicsurveillance pathway. This is the first evidence of c-di-AMP productionand secretion in live bacteria and is the second report of theproduction of this novel di-nucleotide. A single other report assigneddi-adenylate cyclase (DAC) activity to a domain of unknown function(previously DUF147) within the protein DisA of B. subtilis. ¹⁶ Based onthese observations and previous reports pertaining to DisA, it washypothesized that c-di-AMP acts a secondary signaling molecule thatregulates bacterial sporulation. Bio-informatic analysis showed thewidespread presence of the DAC domain in bacteria and archeae, includingpathogenic Staphylococci, Streptococci, Mycobacteria, Chlamydia, andMycoplasma spp.¹⁷ Analysis of the genome of L. monocytogenes revealed asingle gene, Imo2120, containing a predicted DAC domain. This gene ispresent in an operon with the downstream gene Imo2119, a gene of unknownfunction (FIG. 4 a). Attempts to delete the gene Imo2120 using standardtechniques were unsuccessful. Genes containing DAC domains inStreptococci and two species of Mycoplasma have been identified asessential,¹⁸⁻²⁰ supporting a similar indispensable role in L.monocytogenes. However, over-expression of Imo2120 did not affectbacterial growth but did lead to increased CSP activation duringmacrophage infection FIGS. 4 b-c). These results are consistent with DACactivity encoded by the Imo2120 gene, which we have named here dacA.

A transposon insertion in L. monocytogenes Imo0052 was identified in aforward genetic screen for mutants that affect host cell death and IFN-βproduction (Sauer, J. D., et al. (2010). Cell Host Microbe 7, 412).Disruption of Imo0052 resulted in elevated levels of IFN-β compared toinfection with wild-type L. monocytogenes. Sequence analysis identifiedLmo0052 as a homolog of B. subtilis YybT, which was recently shown tohave c-di-AMP phosphodiesterase activity (Rao, F., et al. (2010). J BiolChem 285, 473). Lmo0052, renamed here PdeA, has 50% identity and 74%similarity to YybT and shares the multi-domain structure of YybT withtwo N-terminal transmembrane domains, a PAS signaling domain, a modifiedGGDEF domain, and C-terminal DHH and DHH-associated domains. PurifiedPdeA catalyzed conversion of c-di-AMP to the linear dinucleotide pApA,and purification of truncated constructs showed this activity waslocalized to the DHH/DHHA1 domains, thereby confirming previousbiochemical characterization of homologous proteins (Rao et al., supra).Together these data suggest that PdeA is a c-di-AMP phosphodiesterase.

To define how each of these proteins affects c-di-AMP, we constructed aclean deletion of pdeA and over-expressed the gene. To achieve highlevels of over-expression, pdeA was placed downstream of P_(actA) in thePrfA* G145S background strain, where the promoter is constitutivelyactive at high levels (Ripio, M. T., et al. (1997). J Bacteriol 179,1533). As discussed above, we also manipulated expression of DacA. To dothis, we introduced of a second copy of the dacA gene using anIPTG-inducible integration vector, pLIV2 (Fischetti, V. A. (2006).“Gram-Positive Pathogens”, ASM Press, Washington, D.C.). In this geneticbackground, successful deletion of the chromosomal dacA was accomplishedin the presence of inducer (cΔdacA). Removal of IPTG from these culturesresulted in a conditional dacA mutant (L1, Z., et al. (2005). InfectImmun 73, 5065).

Multiple attempts to measure intracellular levels of c-di-AMP wereunsuccessful. To define how expression of these proteins affectsc-di-AMP metabolism, c-di-AMP secretion by each strain was measured inchemically defined minimal media. As discussed above, ectopicover-expression of bacterial MDRs or DacA resulted in increased levelsof c-di-AMP in the culture supernatant. Both the conditional deletion ofdacA and over-expression of pdeA were predicted to lead to depletion ofc-di-AMP. Indeed, lower levels of c-di-AMP were observed in culturesupernatants of these strains, although it should be noted that cΔdacAmutant reached 50% of the levels of growth of other strains in minimalmedia. The clean deletion of pdeA was predicted to result in high levelsof intracellular c-di-AMP, as has been shown in B. subtilis andStaphylococcus aureus mutants deficient in PdeA homologs (Corrigan, R.M., et al. (2011). PLoS Pathog 7, e1002217; Oppenheimer-Shaanan, Y., etal. (2011). EMBO Rep 12, 594). Surprisingly, PdeA-deficiency did notsignificantly affect levels secreted into the supernatant during brothculture even though increased IFN-β was observed during infection.

As discussed above and in Sauer et al. 2011 (Sauer, J D et al. (2011).Infect Immun 79, 688) c-di-AMP secreted by L. monocytogenes duringinfection leads to Type 1 interferon production. Although pdeA-deficientmutants are predicted to contain high levels of intra-bacterialc-di-AMP, we observed that the amount secreted by these mutants into theculture supernatant was comparable to levels observed with wild-type L.monocytogenes. To address this paradox, we infected murine bonemarrow-derived macrophages and quantified induction of IFN-β by qRT-PCR.PdeA-deficient mutants stimulated nearly 5-fold more IFN-β thanwild-type L. monocytogenes, indicating that more c-di-AMP is secreted inthe host cell cytosol. This intracellular-specific release of c-di-AMPsuggests that some condition within the host cell may trigger c-di-AMPsecretion during infection and is physiologically distinct from thesignal that leads to secretion during in vitro broth growth.

C. Discussion

Active secretion of a specific cyclic di-nucleotide by L. monocytogenesMDR transporters is an unprecedented observation with two importantimplications. First, MDRs are generally recognized to function inconferring resistance to small toxic molecules by active efflux,preventing accumulation of lethal concentrations within the cell. Anumber of instances have described transport of small molecules that arenot toxic,²¹⁻²³ leading to the hypothesis that these transporters haveevolved to transport specific natural substrates as well.²⁴ Theobservations presented show that these proteins play a broaderbiological role beyond general drug resistance, including bacterialsignaling. Second, bacterial signaling nucleotides are generallyconsidered to act within the cell. Here, we show that this molecule maybe exported from the cell. These observations show that c-di-AMP isinvolved in extracellular signaling by L. monocytogenes.

Sensing of conserved and essential microbial molecules by the host is anevolutionary adaptation that maximizes microbial detection with alimited number of germ-line encoded receptors. Given the widespreadpresence of the DAC domain and the important role it plays in bacterialgrowth, this bacterial specific nucleotide is an attractive innateimmune ligand. A number of reports demonstrate that microbe specificnucleotides induce inflammation in the host and, due to immunomodulatoryeffects, enhance protection to bacterial infection.²⁵⁻²⁸ Furthermore,c-di-GMP specifically activates the host IFN pathway when delivered tothe host cytosol.²⁹ Given the widespread appearance of the DAC domain inbacteria, it is likely that c-di-AMP is involved in pathogen recognitionbeyond L. monocytogenes, and, together with c-di-GMP, represent a classof ligands responsible for cytosolic activation of IFN-β by bacteria.

C. References

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D. Materials and Methods 1. Site Directed Mutants of MdrM

The open reading frame of MdrM was amplified from L. monocytogenesstrain 10403S genomic DNA and the stop codon was removed using thefollowing primers: 3′-GAG GAG CAT ATG AAT ATG AAA GCA GCA AGT ACA TCAG-5′ (SEQ ID NO:01) and 3′-GAG GAG CTC GAG TGC TTT TTC CGT TTT AGT AACAAT TG-5′ (SEQ ID NO:02). The resulting fragment was digested with NdeIand XhoI and ligated into similarily digested pET20b. The resulting openreading frame, containing a hexa-histidine tag, was subsequentlyamplified using the following primers: 3′-GAGGAG CGG CCG ATG AAT ATG AAAGCA GCA AGT ACA TC-5′ (SEQ ID NO:03) and 3′-GAGGAG GTC GAC TCA GTG GTGGTG GTG GTG G-5′ (SEQ ID NO:04). The amplification product was digestedusing EagI and SalI and ligated into similarily digested pLIV2 togenerate the IPTG inducible construct pLIV2:MdrMHis6x. The R109A MdrMDNA construct was made using quick change mutagenesis with the followingprimers: 5′-CCA TGC TGA TTG CTG GGG CAA TGG TAC AAG CAA TTG G-3′ (SEQ IDNO:05) and 5′-CCA ATT GCT TGT ACC ATT GCC CCA GCA ATC AGC ATG G-3 (SEQID NO:06)′ with pLIV2:MdrMHis6x as the template DNA. The G154C MdrM DNAconstruct was made using quick change mutagenesis with the followingprimers: 5′-GAA CTT TGC CCC AGC AAT TTG CCC GAC ACT TTC AGG-3′(SEQ IDNO:07) and 5′-CCT GAA AGT GTC GGG CAA ATT GCT GGG GCA AAG TTC-3′ (SEQ IDNO:08) with pLIV2:MdrMHis6x as the template DNA. DNA sequencing wasperformed by the UC Berkeley DNA sequencing. Mutant plasmid wastransformed into chemically Z-competent (Zymo Research) SM10 E. colicells and conjugated with the mdrM-L. monocytogenes strain.

2. Intracellular Growth Curves

Bacterial growth in bone marrow derived macrophages was performed asdescribed previously (S1). Briefly, BMMs (3×106 cells/dish) were platedin a monolayer on 12×1 mm round cover slips in 60 mm round petri dishesthe night before infection. Bacteria were grown overnight at 30° C. inBHI. When appropriate, bacteria were grown overnight with IPTG (1 mM).In the morning bacteria were pelleted by centrifugation and washed 3times with PBS. Macrophages were infected with 2×105 bacteria.Infections were washed 3× with PBS at 30 minutes post infections andgentamycin (50 μg/mL) was added at 1 hour post infection. Cover slipswere removed and placed into sterile MQ water to lyse macrophages.Various dilutions were plated to determine the number of colony formingunits.

3. DacA Inducible L. monocytogenes Strain

An IPTG inducible plasmid containing the open reading frame (ORF) ofdacA (Imo2120) was integrated into the L. monocytogenes chromosome. TheORF of dacA was amplified using the following primers: 5′-GAG GAG CGGCCG ATG GAT TTT TCC AAT ATG TCG ATA TTG-3′ (SEQ ID NO:09) and 5′-GAG GAGGTC GAC ATT TAA AAT TCG ATC CAT CAT TCG CT-3′ (SEQ ID NO:10). The PCRamplification product was then digested with EagI and SalI and ligatedwith similarly digest pLIV2 plasmid. The plasmid was then conjugatedinto L. monocytogenes using E. coli SM10. DNA sequencing was performedby the UC Berkeley DNA sequencing.

4. Fractionation of L. monocytogenes Culture Supernatants

Various strains of L. monocytogenes were cultured overnight shaking at37° C. in brain heart infusion (BHI) media. The following morning,overnight cultures were used to inoculate 50 mL of BHI media(OD600=0.02), which were subsequently grown with shaking at 37° C. untilOD600=0.5. Cells were then pelleted by centrifugation and resuspended in50 mL of chemically defined minimal media (S2). Cultures were grown at37° C., shaking for 19 hours followed by centrifugation to removebacteria. The supernatant was subsequently filtered through 0.4 μmnitrocellulose membrane filter (Millipore) and the pH of thesupernatants was adjusted to 4.0 with concentrated HCl. Supernatantswere then applied to Sep-Pak columns (Waters Corp., 6 cc, 500 mg C18) bygravity. The column was subsequently washed in succession with 5 mL eachof 0.1% (w/v) trichloroacetic acid (TCA), 50% MeOH mixed with 0.1% TCA,and MeOH. Each wash was collected as a separate fraction and dried usinga SpeedVac concentrator. The resulting pellet was resuspended in 1 mL ofwater and assayed for IFN-β stimulatory activity. Active fractions werefurther fractionated using reversed-phase high performance liquidchromatography (RP-HPLC) on an Agilent 1100 LC system. A fraction of theactive sample (100 μL) was loaded onto a Waters Nova-Pak C18 column (4μm, 3.9×150 mm) equilibrated with 0.1% formic acid (Solvent A). Thecolumn was developed at 0.5 mL/minute with an initial 5 minute wash with100% Solvent A, a gradient of 0-50% MeOH over 40 minutes, a gradientfrom 50-100% MeOH over 5 minutes, followed by 100% MeOH for 3 minutesand a return to 100% water. Fractions (0.5 mL) were collected over theentire run. Each fraction was dried using a SpeedVac, resuspended in 50μL of water, and tested for IFN-β stimulating activity in bone marrowderived macrophages.

5. Purification and Digestion of L. monocytogenes Peptidoglycan

L. monocytogenes grown overnight in BHI was used to inoculate 1 Lculture of BHI to an optical density of 0.02. Cells grown at 37° C. withshaking to an optical density of 1 were harvested by centrifugation.Bacteria were resuspended in PBS with 5% SDS and boiled for 30 minutes.Cells were separated by centrifugation and then washed 2 times withwater and then run through a French press 3 times at 14000 psi. Cellsthen were resuspended in PBS and bead beat for 10 minutes andsubsequently separated by centrifugation. Pellets from centrifugationwere resuspended in 4% boiling SDS for 30 minutes. Sample was cooled andcell wall was pelleted by centrifugation. Supernatant was removed andpellet resuspended in 4% SDS and boiled for 15 minutes. Cell wall wasthen washed with hot water, 0.1% Triton, and five more times with hotwater. Finally, purified cell wall was resuspended in mutanolysin buffer(50 mM MES pH 5.9, 1 mM MgCl₂). Cell wall (15 mg) was digested withmutanolysin (50 units, Sigma Aldrich, Streptomyces globisporus) in thepresence and absence of DNAse (5 Kunitz units, Qiagen). IFN-β inducingactivity of these samples was assessed using the IFN-β bioassay.Attempts to use digitonin delivery with DNA were unsuccessful witheither double stranded DNA oligonucleotides or digested bacterial cellwall. As such, delivery of samples to the macrophage cytosol wasperformed using Lipofectamine 2000 (Invitrogen) according to themanufacturer's protocol.

6. Ion Exchange Pull-Down

To characterize the charge of the active component secreted by L.monocytogenes, active fractions from HPLC purification of themarRsupernatant were incubated with cation-exchange resin (SP650M,Toyopearl) or anionexchange resin (Q650M, Toyopear) for 30 minutes withagitation at room temperature. Resin was removed by centrifugation andthe activity remaining in the supernatant was measure by IFN-βbio-assay.

7. IFN-β Bio-Assay

Mouse bone marrow from 6-8 week old female mice was differentiated tobone marrow derived macrophages (BMM) as described previously (S3).Interferon responsive ISRE-L929 cells were cultured in ISRE media (DMEM,2 mM glutamine, 1 mM pyruvate, 10% heat inactivated FBS, andpenicillin-streptomycin). Induction of type-I interferon was assessedusing BMMs plated in 96-well flat bottom tissue culture treated plates(105 cells/well) a minimum of 12 hours prior to use in BMM media (DMEM,2 mM glutamine, 1 mM pyruvate, 10% CSF from 3T3 cells, and 20% heatinactivated FBS). Samples (10 μL) of L. monocytogenes supernatants, HPLCfractions, and cyclic di-AMP standard (Biolog Life Sciences Institute,Denmark) were mixed with a 10× volume of digitonin permeabilizationsolution (50 mM HEPES pH 7.0, 100 mM KCl, 3 mM MgCl2, 0.1 mM DTT, 85 mMSucrose, 0.2% BSA, 1 mM ATP, 0.1 mM GTP, ±10 μg/mL Digitonin) (S4).Media was aspirated from the cells and replaced with 50 μL each samplemixture. Cells were incubated for 30 minutes at 37° C. Wells were againaspirated and fresh BMDM media (50 μL/well) was added. At 4 hours postinitial addition of sample, supernatants were removed and applied invarious dilutions to the interferon responsive ISRE-L929 cells (5×104cells/well) in white, 96-well, tissue culture treated plates (ThermoScientific Nunc). Cells were incubated for 4 hours, media aspirated, and40 μL of TNT lysis buffer (20 mM Tris, 100 mM NaCl, 1% triton, pH 8.0)was added to each well. Finally, 40 μL of luciferase substrate solution(20 mM Tricine, 2.67 mM MgSO₄.7H₂O, 0.1 mM EDTA, 33.3 mM DTT, 530 μMATP, 270 μM acetyl CoA lithium salt, 470 μM luciferin, 5 mM NaOH, 265 μMmagnesium carbonate hydroxide) was added to each well and luminescencewas measured using a VICTOR3 luminometer (PerkinElmer).

8. Liquid Chromatography Mass Spectrometry (LC-MS) Analysis

Listeria monocytogenes fractions were analyzed using an Agilent 1200series liquid chromatograph (LC; Santa Clara, Calif.) connected in-linewith an LTQ Orbitrap XL hybrid mass spectrometer equipped with an IonMax electrospray ionization source (ESI; Thermo Fisher Scientific,Waltham, Mass.). Acetonitrile (Fisher Optima grade, 99.9%) and formicacid (Pierce, 1 mL ampules, 99+%) purchased from Fisher Scientific(Pittsburgh, Pa.), and water purified to a resistivity of 18.2 MΩ·cm (at25° C.) using a Milli-Q Gradient ultrapure water purification system(Millipore, Billerica, Mass.), were used to prepare mobile phasesolvents for liquid chromatography. The LC was equipped with C8 guard(Poroshell 300SB-C8, 5 μm, 12.5×2.1 mm, Agilent) and analytical (75×0.5mm) columns. Solvent A was 0.1% formic acid/99.9% water and solvent Bwas 0.1% formic acid/99.9% acetonitrile (v/v). Sample solutionscontained in 0.3 mL polypropylene snap-top vials sealed with rubbersepta caps (Wheaton Science, Millville, N.J.) were loaded into theAgilent 1200 autosampler compartment prior to analysis. A 50 μLinjection volume was used for each sample.

Following sample injection, analyte trapping was performed for 5 minwith 99.5% A at a flow rate of 90 μL/min. The elution program consistedof a linear gradient from 5% to 95% B over 27 min, isocratic conditionsat 95% B for 10 min, a linear gradient to 0.5% B over 1 min, and thenisocratic conditions at 0.5% B for 16 min, at a flow rate of 90 μL/min.The column and sample compartment were maintained at 35° C. and 10° C.,respectively. Solvent (Milli-Q water) blanks were run between samples,and the autosampler injection needle was rinsed with Milli-Q water aftereach sample injection, to avoid cross-contamination between samples. Theconnections between the LC column exit and the ESI probe of the massspectrometer were made using PEEK tubing (0.005″ i.d.× 1/16″ o.d.,Western Analytical, Lake Elsinore, Calif.). External mass calibrationwas performed prior to analysis using the standard LTQ calibrationmixture containing caffeine, the peptide MRFA, and Ultramark 1621dissolved in 51% acetonitrile/25% methanol/23% water/1% acetic acidsolution (v/v). The ESI source parameters were as follows: ion transfercapillary temperature 275° C., normalized sheath gas (nitrogen) flowrate 25%, ESI voltage 2.0 kV, ion transfer capillary voltage 49 V, andtube lens voltage 120V. Full scan mass spectra were recorded in thepositive ion mode over the range m/z=100 to 1500 using the Orbitrap massanalyzer, in profile format, with a full MS automatic gain controltarget setting of 5×105 charges and a resolution setting of 6×104 (atm/z=400, FWHM). In the data-dependent mode, the most intense ionmeasured from each full scan mass spectrum exceeding an intensitythreshold of 15,000 counts was selected for tandem mass spectrometry(MS/MS) analysis. MS/MS spectra were acquired using the Orbitrap massanalyzer, in profile format, with a resolution setting of 1.5×104 (atm/z=400, FWHM), using collisionally activated dissociation (CAD) with anisolation width of 2 m/z units, a normalized collision energy of 28%,and a default charge state of 1+. To avoid the occurrence of redundantMS/MS measurements, real-time dynamic exclusion was enabled to precludere-selection of previously analyzed precursor ions using a repeat countof one, a repeat duration of 5 s, a maximum exclusion list size of 100different precursor ions, an exclusion duration of 180 s, and anexclusion width of 1.5 m/z units.

Mass spectra and MS/MS spectra were processed using Xcalibur software(version 4.1, Thermo). ChemBioDraw Ultra software (version 11.0.1,CambridgeSoft, Cambridge, Mass.) was used to draw and calculate theexact masses of candidate chemical structures for comparison withmeasured masses. To quantify c-di-AMP in L. monocytogenes supernatants,the four fractions eluted from HPLC purification of the nucleotidesurrounding the peak of activity were pooled for each strain andanalyzed by LC-MS. The observed peak for ion 659.11 m/z was integratedusing Xcalibur software. The concentration of the nucleotide wasdetermined by comparing the integrated peak area to a calibration curvegenerated using synthetic c-di-AMP samples of known concentrationanalyzed in a similar way.

9. IFN-β Induction in Various Host Strains

BMMs were differentiated as described previously (S3). For mavs−/−macrophages, heterozygous mice were used as a control. WT and mutantBMMs were plated in 24-well plates (5×106 cells/well) the evening priorto use. For infections, 2×106 bacteria from 30° C. overnight cultureswere washed with PBS and added to each well containing 500 μL of BMMmedia. For c-di-AMP treatment, synthetic standard was dissolved indigitonin solution to a final concentration of 3 μM. Media was aspiratedfrom cells and replaced with 200 μL of c-di-AMP/digitonin mix. From thispoint on infections and c-di-AMP samples were treated the same. At 30minutes wells were aspirated and replaced with fresh BMM media. At 1hour post infection gentamycin (50 μg/mL) was added. For LPS treatment,100 ng of sonicated LPS stock solution (50 μg/mL) was added toappropriate wells containing 500 μL of BMM media. Supernatants from allwells were collected and analyzed by ISRE L929 bioassay for IFN-βactivity at 6 hours.

10. Supporting References

-   S1. D. A. Portnoy, P. S. Jacks, D. J. Hinrichs, Role of hemolysin    for the intracellular growth of Listeria monocytogenes. J. Exp. Med.    167, 1459 (1988).-   S2. L. Phan-Thanh, T. Gormon, A chemically defined minimal medium    for the optimal culture of Listeria. Int. J. Food Microbiol. 35, 91    (1997).-   S3. J. H. Leber et al., Distinct TLR- and NLR-mediated    transcriptional responses to an intracellular pathogen. PLoS Pathog.    4, e6 (2008).-   S4. S. E. Girardin et al., Nod1 detects a unique muropeptide from    gram-negative bacterial peptidoglycan. Science 300, 1584 (2003).

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

That which is claimed is:
 1. A method of modulating type-I interferon production in a eukaryotic cell, the method comprising: introducing into the cell a bacterium that increases cytosolic cyclic di-adenosine monophosphate (c-di-AMP) activity in the eukaryotic cell in a manner sufficient to increase type-I interferon production in the cell, wherein the bacterium is a mutant that has, as compared to its corresponding wild-type bacterium, at least one of: (i) enhanced di-adenylate cyclase activity; (ii) enhanced secretion of di-adenylate cyclase; and (iii) reduced c-di-AMP phosphodiesterase activity.
 2. The method according to claim 1, wherein the bacterium is a Listeria bacterium.
 3. The method according to claim 2, wherein the Listeria bacterium is a mutant that secretes enhanced amounts of c-di-AMP as compared to its corresponding wild-type bacterium.
 4. The method according to claim 3, wherein the Listeria bacterium is a mutant that has enhanced di-adenylate cyclase activity as compared to its corresponding wild-type bacterium.
 5. The method according to claim 4, wherein the Listeria bacterium overexpresses Imo2120 or a mutant thereof.
 6. The method according to claim 3, wherein the Listeria bacterium is a mutant that has reduced c-di-AMP phosphodiesterase activity as compared to its corresponding wild-type bacterium.
 7. The method according to claim 6, wherein the Listeria bacterium comprises an Imo0052 mutation.
 8. The method according to claim 2, wherein the bacterium is a mutant with enhanced secretion of di-adenylate cyclase as compared to its corresponding wild-type bacterium.
 9. The method according to claim 8, wherein the di-adenylate cyclase is Imo2120 or a mutant thereof.
 10. The method according to claim 1, wherein the method comprises enhancing cytosolic di-adenylate cyclase activity in the cell.
 11. The method according to claim 10, wherein the method comprises overexpressing a polypeptide having di-adenylate cyclase activity.
 12. The method according to claim 1, wherein the cell is a macrophage.
 13. The method according to claim 1, wherein the type-I interferon is interferon-β.
 14. The method according to claim 1, wherein the cell is in vitro.
 15. A mutant Listeria bacterium comprising a mutation which enhances secretion of a compound selected from the group consisting of: cytosolic di-adenylate cyclase or c-di-AMP phosphodiesterase and combinations thereof; as compared to its corresponding wild-type control.
 16. A method for modulating interferon-β production in a mammalian subject, the method comprising: administering to the mammalian subject an effective amount of a Listeria bacterium according to claim
 15. 17. The Listeria bacterium according to claim 15, wherein the mutation enhances di-adenylate cyclase activity in the bacterium.
 18. The Listeria bacterium according to claim 17, wherein the mutation is an Imo2120 mutation that enhances the expression of Imo2120.
 19. The Listeria bacterium according to claim 15, wherein the mutation enhances secretion of cytosolic di-adenylate cyclase.
 20. The Listeria bacterium according to claim 19, wherein the bacteria overexpresses Imo2120 or a mutant thereof.
 21. The Listeria bacterium according to claim 15, wherein the Listeria bacterium is Listeria monocytogenes.
 22. The Listeria bacterium according to claim 15, wherein the Listeria bacterium is attenuated.
 23. The Listeria bacterium according to claim 17, wherein the Listeria bacterium increases interferon-β production in macrophages as compared to its corresponding wild-type control.
 24. The Listeria bacterium according to claim 15, wherein the bacterium comprises a heterologous nucleic acid.
 25. The Listeria bacterium according to claim 24, wherein the heterologous nucleic acid is integrated.
 26. The Listeria bacterium according to claim 24, wherein the heterologous nucleic acid encodes at least one product.
 27. The Listeria bacterium according to claim 26, wherein the at least one product is an antigen. 